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C . . . . o ‘ . .. . xi .. l ' meAev Micnigan State University This is to certify that the dissertation entitled SOMATIC CELL NUCLEAR TRANSFER IN ZEBRAFISH presented by KANNIKA SIRIPA'ITARAPRAVAT has been accepted towards fulfillment of the requirements for the Doctoral degree in Comparative Medicine and Integrative Biologx (njjor Professor’s Signature '5 - G -— 2010 Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:lProj/AccalPresIClRCIDateDue.indd SOMATIC CELL NUCLEAR TRANSFER lN ZEBRAFISH BY Kannika Siripattarapravat A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology 2010 ABSTRACT SOMATIC CELI. NUCLEAR TRANSFER IN ZEBRAFISH BY Kannika Siripattarapravat Zebrafish (Danio rerio) have been recognized as one of the best model organisms for the study of developmental biology and human diseases. As of yet, the utilization of zebrafish has not reached its promise. To fulfill this potential, a methodology to generate conditional knock—in/ -out must be developed. Somatic cell nuclear transfer (SCNT) is a potential approach to produce genetically-modified zebrafish. This can be accomplished by transferring gene~targeted cells into enucleated eggs. The primary focus of this dissertation is to improve the efficiency of SCNT. Subsequently, the use of SCNT technology may be extended to enhance the use of zebrafish as a vertebrate animal model. Three important parameters have been characterized and optimized to meet such a goal: recipient eggs, nuclear transfer technique, and cultured donor cells. While the zebrafish cloning technique has been published, it is highly inefficient. Moreover, the existing protocol is difficult to replicate, likely due to poor characterization of zebrafish egg physiology at the time of nuclear transfer. We have demonstrated that, following egg activation, eggs undergo dynamic changes in cell cycle stages and that is likely to affect cloning efficiency. To improve upon this, we implemented a technique in which recipient eggs can be maintained in vitro at metaphase II of meiosis (Mil) stage in Chinook salmon ovarian fluid. This should provide a uniform source of recipient eggs for SCNT. Accordingly, W9- have developed a reliable SCNT protocol that overcomes the challenge of using zebrafish MII eggs with intact chorion as recipient cells for SCNT, by using laser-assisted inactivation of egg genome and micropyle for transfer of the nucleus. This technique has been validated by using phenotypic screening, karyotyping, and genotyping of cloned zebrafish produced. Cloned zebrafish are normal healthy individuals, and go on to produce thousands of healthy offspring. The SCNT technique can be used to produce clones from the major strains of zebrafish used in the research community. Additionally, we have showed that zebrafish SCNT can be used to investigate the influence of donor cell sources on cloning efficiency. By using transgenic fish that express tissue specific green fluorescence protein (GFP) as sources of donor cells, we have found that the type of donor cells used in SCNT influences the developmental capacity of the cloned fish from the blastula stage up to 4 days. In parallel, we have done extensive work to optimize the in vitro culture conditions for zebrafish cells, and described new cell culture and DNA transfection protocols for cultured cells. We explored the possibility of increasing SCNT efficiency by modifying the donor nuclei using histone deacetylase (HDAC) inhibitors. Our SCNT model can be further implemented in combination with existing technology to facilitate gene knock-in/ -out experiments in zebrafish. The ultimate goal is to enhance its prominent role as an animal model for human diseases. Copyfightby KANNIKA SIRIPATTARAPRAVAT 2010 DEDICATION To my beloved family: my parents, sisters and brother. To my father, Pichit Siripattarapravat, who has always believed the best thing he can give me is his support toward achieving the highest level of education. As a consequence, this has been my primary motivation for earning a PhD. To my mother, Viyada Siripattarapravat, who has always given me strength from her unreserved love and encouragement. To my sisters and brother whom always have stood by me. ACKNOWLEDGMENTS I would like to express my sincere gratitude to many individuals who have supported and encouraged me toward finishing this dissertation. I am most in debt to the members of my guidance committee: advisor Dr. Jose Cibelli, who has given me academic freedom with great faith and patience, whose passion in science has always inspired me, and friendship that has helped carry me through; Dr. C.C. Chang, who has never hesitated to teach me all things he knows; Dr. Gloria Perez, who has challenged me with many questions and supported me in every way possible; Dr. Weiming Li, who has always encouraged me to perceive things broadly; and Dr. Paul Collodi, who has granted me his expertise in zebrafish and generously provided fish and reagents used in experiments. I am likewise grateful to the Royal Thai government and Kasetsart University for the studentship, and the Comparative Medicine and Integrative Biology (CMIB) program in the College of Veterinary Medicine for admitting me to their graduate program. I give my sincere gratitude to the CMlB program. Special thanks go to Dr. Vilma Yuzbasiyan- Gurkan, the program director, who always has believed in me and enabled the opportunity to study at Michigan State University; much appreciation to Dr. Victoria Hoelzer-Maddox, who kindly assisted and made things easier during my study; and most of all the warm welcome, great environment, and life—time friends that include Donna Housley, Chidozie Amuzie, Manish Neupane, and Tuddow Thaiwong. vi This dissertation could not be completed without help and suggestions from the members of Cellular Reprogramming Laboratory. Specifically, I must give the credit to: Boonya Pinmee, the other fish cloner, who has been working so hard and supported me in every way; Angela Busta, Ashley Mckay and Shashanka Murthy for being reliable fish fellows; Ramon Rodriguez Lopez, for his generous help on immunostaining, imaging, and innovative ideas; Dr. Steve Suhr, for infecting me with his limitless ideas for experiments; and Dr. EunAh Chang, for great discussion, patient support, and for being there for me since the very first day I started in the lab. I also highly appreciate both present and past lab members whom have provided such a wonderful working environment all these years. I am grateful for Dr. Patrick Venta who both generously taught and helped me on the genetic analysis. I am very pleased to have the opportunity to consult with Drs. Scot Wolfe, Nathan Lawson, and Ten-Tsao Wong on the generation of gene-targeted fish. I also value the time I spent with former fish cloner Dr. Ki-Young Lee, who kindly taught me all of his special techniques. I thank Drs. Juan Pedro Steibel, Sungworn Ngudgratoke, and Juan David Munoz for their generous support on statistical analysis. I cannot give enough thanks to Michigan Department of Natural Resources, especially the egg takers at little Manistee weir for donating the Chinook salmon ovarian fluid. Last but not least, this project is funded by the endowed research fund - College of Veterinary Medicine, the Michigan State University Experiment Station, the office of the Vice President for Research and Graduate Studies, the Michigan State University foundation, and the Naylor Family Foundation. vii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... x LIST OF FIGURES ...................................................................................................................... xi LIST OF ABBREVIATIONS ..................................................................................................... xiii INTRODUCTION ....................................................................................................................... 1 Rationale and specific aims ............................................................................. 1 Summary of chapters ....................................................................................... 3 Significant .......................................................................................................... 4 CHAPTER 1: LITERATURE REVIEW .......................................................................................... 6 Zebrafish as a model organism ........................................................................ 6 Methods for transgenesis, forward, and reverse genetics in zebrafish ......................................................................................................... 8 The recipient cell for nuclear transfer .......................................................... 22 CHAPTER 2: THE DONOR CELLS FOR ZEBRAFISH SOMATIC CELL NUCLEAR TRANSFER: OPTIMIZATION OF IN VITRO CULTURE CONDITIONS, DNA TRANSFECT ION, AND CHEMICALLY-ASSITED EPIGENETIC MODIFICATIONS........................................................30 Abstract ............................................................................................................ 30 Introduction ..................................................................................................... 30 Result ................................................................................................................. 33 Discussion ......................................................................................................... 39 Methods ............................................................................................................ 42 CHAPTER 3: CHARACTERIZATION AND IN VITRO CONTROL OF MPF ACTIVITY IN ZEBRAFISH EGGS .................................................................................................................... 55 Abstract ............................................................................................................. 55 Introduction ..................................................................................................... 56 Result ................................................................................................................. 60 Discussion ......................................................................................................... 65 Methods ............................................................................................................ 68 CHAPTER 4: SOMATIC CELL NUCLEAR TRANSFER IN ZEBRAFISH 80 Abstract ............................................................................................................. 80 Introduction ..................................................................................................... 80 Result ................................................................................................................ 81 Discussion ........................................................................................................ 84 Methods ........................................................................................................... 85 viii CHAPTER 5: INFLUENCE OF DONOR NUCLEUS SOURCE IN THE OUTCOME OF ZEBRAFISH CLONING PROCEDURES .................................................................................... 99 Abstract ............................................................................................................. 99 Introduction ..................................................................................................... 99 Result ............................................................................................................... 102 Discussion ....................................................................................................... 105 Methods .......................................................................................................... 107 CONCLUSION AND FUTURE DIRECTIONS ......................................................................... 113 REFERENCES ......................................................................................................................... 119 LIST OF TABLES Table 1 Summary of the techniques for transgenesis and mutagenesis studies ............. 21 Table 2 Summary of nuclear transfer in frogs and fish ........................................................ 26 Table 3 Genotyping by SN Ps .................................................................................................... 93 Table 4 Genotyping results ...................................................................................................... 94 Table 5 Development of cloned embryos .............................................................................. 96 Table 6 Efficiency of zebrafish SCNT from GFP+ donor cells of different lineages ......... 111 LIST OF FIGURES (Images in this dissertation are presented in colors) Figure 1 Effect of PBS, TS and EE on cell growth in DMEM ................................................. 46 Figure 2 Comparison of cell growth in DMEMs and K-NACs ............................................... 47 Figure 3 Comparison of cell growth in DMEMs and D-NACs usmg different FBS concentrations ........................................................................................................................... 48 Figure 4 Comparison of cell growth in D-NACs and K-NACs media .................................... 49 Figure 5 Telomerase activity by TRAP assay .......................................................................... 50 Figure 6 Expression of Vimentin in culture cells ................................................................... 51 Figure 7 DNA uptake rates of zebrafish cells using liposome-mediated transfection reagents or electroporation ..................................................................................................... 52 Figure 8 Toxic of VPA in zebrafish embryos ........................................................................... 53 Figure 9 Level of histone acetylation in cultured cells ......................................................... 54 Figure 10 Morphological and molecular changes during parthenogenetic activation of zebrafish eggs ............................................................................................................................. 72 Figure 11 MPF actiVity in eggs activated either by fertilization or parthenogeneSis ....... 73 Figure 12 Nuclear staining of fertilized eggs ......................................................................... 74 Figure 13 In vitro fertilization rates of eggs aged in CSOF ................................................... 75 Figure 14 MPF activity of in vitro aged eggs in either CSOF or H-BSA ............................... 76 Figure 15 Pictures of arrested matured aged eggs in CSOF, H-BSA, 75 uM M6132 in H-BSA, and 10 mM caffeine in H-BSA ................................................................................. 77 Figure 16 Nuclear staining of parthenogenetic embryos .................................................... 78 Figure 17 MPF activity in matured eggs aging in vitro ......................................................... 79 Figure 18 Protocol for SCNT ..................................................................................................... 90 xi Figure 19 Recipient eggs ........................................................................................................... 91 Figure 20 Phenotype of cloned zebrafish and its offspring ................................................. 92 Figure 21 Abnormalities observed in cloned embryos ......................................................... 97 Figure 22 Karyotyping and genotyping of cloned fish .......................................................... 98 Figure 23 GFP+ donor embryos and offspring of cloned fish ............................................. 110 Figure 24 Developmental rate of cloned fish of GFP+ donor cells from different zebrafish lines, included all normal and abnormal embryos ............................................. 112 Figure 25 Possible approaches to generate knock-in/—out zebrafish ............................... 118 xii AB AF A2P BAPTA Cdc2p ClnB CSOF DMEM DNA deT EB EE ENU ES cells ET FBS GFP GR H-BSA HDAC hpf LIST OF ABBREVIATIONS Zebrafish strain AB Adult caudal fin fibroblasts L-ascorbic acid 2-phosphate sesquimagnesium salt 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid Phosphorylated cell division cycle 2 (Cyclin dependent kinase) Cyclin B Chinook salmon ovarian fluid Dulbecco’s Modified Eagle Medium Deoxyribonucleic acid day post-nuclear transfer Zebrafish embryo at 90% epiboly stage Zebrafish embryonic extracts N-Ethyl-N-Nitrosourea Embryonic stem cells Embryonic tailbud Fetal bovine serum Green fluorescence protein Zebrafish embryo at germ ring stage Hank’s balance salt solution with 0.5% bovine serum albumin Histone deacetylase hour post-fertilization xiii hpC hpNT HR H2AzGFP IVF MII MD M PF NAC pA PBS PCR RACE RNA SA SCNT SD SNP TAB TS TSA hour post-collection hour post-nuclear transfer Homologous recombination Histone H2A tagged with GFP In vitro fertilization Keratinocyte SFM mature-arrested egg at metaphase ll of meiosis Morpholinos Maturation promoting factor N-Acetyl-L-Cysteine Polyadenylation signal Phosphate buffered saline Polymerase chain reaction Random amplification of complementary DNA ends Ribonucleic acid Splice acceptor Somatic cell nuclear transfer Splice donor Single-nucleotide polymorphism Outcrossing Tuebingen and AB strains Trout serum Trichostatin A xiv -- m--_ Tu Tu LF VPA ZFN Zebrafish strain tuebingen Zebrafish strain tuebingen —— long fin Valproic acid Wild type zebrafish Zinc finger nuclease XV INTRODUCTION During embryonic development, cells get progressively committed to a specific tissue without taking a reverse path or switching between cell lineages (de- differentiation or trans-differentiation, respectively). However, with somatic cell nuclear transfer—cloning (Gurdon and Uehlinger, 1966; Wilmut et al., 1997), the committed cells can be reprogrammed to reacquire the epigenetic signature of a pluripotent cell. The donor cell subjected to somatic cell nuclear transfer will regain its ability to self-renew and differentiate into every cell type, ultimately developing into a whole organism in a process known as nuclear reprogramming. The results of nuclear reprogramming experiments have confirmed the hypothesis that as a cell differentiates its epigenetic status changes while its genomic DNA remains unaltered. The nuclear reprogramming process involves substantial chromatin remodeling including, but not limited to, histone methylation, acetylation and DNA demethylation, culminating with alteration of gene expression pattern, characteristic of tissue-specific ones, to that of an embryonic cell. Since this is probably the most challenging adaptive assignment fora cell, it is not successful in most cases, accounting for the low efficiency of cloned animal production, approximately 1—5%, across species (Cibelli, 2007). A few of the factors that can influence the success of nuclear reprogramming include factors present in the oocyte, the type of somatic cells used as nuclear donors and the in vitro micro-manipulation of both donor and recipient cells. Due to its many favorable characteristics, the zebrafish is a widely used model for studies of human diseases and developmental biology. In addition to being phylogenetically closer to humans in comparison to flies and worms, the zebrafish also possesses a short generation interval, high fecundity, transparent embryos, low husbandry cost and amenability to large—scale phenotypic screening. In addition, zebrafish share organ similarities to those of mammals, and mutant phenotypes that resemble human genetic diseases. In comparison to the mouse-model, however, homologous recombination has not been successfully implemented in zebrafish, in part, due to the lack of a robust system for derivation of embryonic stem cells - a practical approach for generating knock—in/ ~out mice. To utilize zebrafish model systems at parity with more labor- intensive but more common rodent model systems, a reliable and simple method for reverse genetics is necessary. Up until now, only one group has reported the production of germline-competent embryonic stem cells with the capacity to undergo gene targeting in zebrafish (Fan et al., 2004b; Ma et al., 2001); however nearly a decade after their publication, there is no report yet on the generation of germline-competent founder fish using the aforementioned approach. Somatic cell nuclear transfer (SCNT) has the potential to become the method of choice for germ-line genetic modification in zebrafish. The first successful report of cloned zebrafish was published in 2002 using cultured cells derived from embryos at S- 15 somite stages (Lee et al., 2002). However, the efficiency of cloned fish production over the total number of eggs manipulated has remained at 2% or less. Since the publication, no cloned fish has been produced by the described technique. In this dissertation, current approaches for genetic modification of zebrafish including the generation of knock-in and knock-out fish are discussed. A novel approach for somatic cell nuclear transfer in zebrafish is described. Also the major parameters that influence the success of cloning such as the condition of donor nuclei and recipient oocytes including pretreatment of oocytes with compounds to maintain and/or enhance their reprogramming capacity are investigated. Finally, the use of donor cells isolated from different cell lineages is explored. Chapter 1 is a literature review where we justify zebrafish use as a model organism. We review current approaches and tools for transgenesis and mutagenesis. More specifically to SCNT, we discuss the different recipient cells used in the procedure. Chapter 2 focuses on in vitro handling of zebrafish cultured donor-cells and the optimization of in vitro culture conditions and DNA transfection. This chapter includes culture medium and supplements for zebrafish cells, major characteristics of the cultured cells, and transfection efficiency. I describe our attempts to alter the epigenetic configuration of donor nuclei to facilitate nuclear reprogramming. The epigenetic status of zebrafish cells derived from both embryo and adult tissue is also addressed. Trichostatin A and Valproic acid, the small molecules, were evaluated for their effects on epigenetic modification to improve nuclear reprogramming efficiency. Chapter 3 focuses on characterization of maturation promoting factor as well as modulation of its activity in zebrafish eggs. An ideal cell cycle stage of the recipient eg for somatic cell nuclear transfer is described. This chapter also addresses the differences of zebrafish eggs before and after activation and ultimately provides a key success of the cloning technique described in Chapter 4. Chapter 4 describes a novel methodology for somatic cell nuclear transfer in zebrafish that is able to produce cloned fish in a routine basis. The approach employs non-activated recipient eggs, laser-firing enucleation, and nuclear transfer through a micropyle. This novel approach overcomes challenges of the first cloning technique reported that include extending time of manipulation, ensuring removal of egg genome, reducing technical difficulties due to handling de-chorinated eggs and fragile reconstructed embryos. Chapter 5 describes the type of donor cell that provides high efficiency of SCNT in zebrafish. In this chapter we address the hypothesis that the capacity of a cell to be reprogrammed depends upon the type of cell used as a nuclear donor. Zebrafish SCNT efficiency was evaluated using different donor cells isolated from 5 different tissues. In doing so, the rate of cloned fish production from embryonic donors derived from 5 different transgenic strains were compared; HGn62A—skin, HGn28A—skin, HGn8E—heart, HGZlC—fin and notochord, and HGn30A-hatch gland. The last chapter is the conclusion and future direction of our work. We summarize the work done by us and others, and discuss the possible next steps for this area of study. The multitude‘of favorable attributes held by zebrafish, coupled with the recent successful nuclear transfer using long-term-cultured cells make this vertebrate the prime candidate for gene loss-of-function studies. The work described in this dissertation includes the development of an efficient system for nuclear transfer of zebrafish using somatic cells, a technology that may enable the creation of gene knock— out/ —in models. Taken together the studies help provide a more clear direction that should be taken in working to improve efficiency of nuclear transfer cloning in zebrafish. CHAPTER 1 LITERATURE REVIEW A. Zebrafish as a model organism Zebrafish, Danio rerio, belong to the Cyprinidae family, the same as carps and minnows. Adult zebrafish are relatively small fresh water fish, approximately 3-4 centimeters in length, allowing them to be kept in a simple aquarium system in almost any laboratory. The embryos are transparent and develop ex vivo, making it possible to study them in detail from fertilization to hatching. Zebrafish are native to the tropical climate of Asian countries, and were first identified in India (Engeszer et al., 2007). They were extensively promoted as a model organism by George Streisinger at the University of Oregon and quickly embraced by the world’s community of developmental biologists. As the zebrafish model gained in popularity, the zebrafish research community created the 'Zebrafish Information Network’ (ZFIN) that contains a variety of essential information on this model organism. There are many fish strains that are utilized in the research community, the more popular ones being Tuebingen, AB, and wildtype. As a laboratory animal the zebrafish possesses many distinct advantages over the mouse and can be considered one of the best animal models for developmental studies, particularly if gene loss of function studies were available. Zebrafish represents a vertebrate that can serves as a convenient, relatively inexpensive, and useful model . for the study of normal and pathological animal development, physiology, aging, cell death, and disease (Beis and Stainier, 2006; Berghmans et al., 2005; Kishi S, 2002; Pyati et al., 2007). Desirable characteristics possessed by zebrafish include its fecundity, external fertilization, rapid embryonic development, and a short generation interval (Niisslein-Volhard and Dahm, 2002; Zon, 1999). Furthermore, zebrafish eggs are large and transparent, facilitating DNA injection, cell labeling, and transplantation experiments (Lee et al., 2002). Despite the lack of a reliable reverse—genetic system, the aforementioned beneficial traits found in zebrafish have promoted the extension of large-scale mutagenesis screening of this organism. Taken together, these features make zebrafish a great complement to D.melanogaster and Celegans for developmental biology studies (Amsterdam et al., 2004; Driever et al., 1996; Haffter P, 1996) and become the widely used vertebrate. The overarching theme of this dissertation is the development of new tools and reagents that can make zebrafish a more powerful animal model to understand human disease. One of the current shortcomings of the zebrafish as a model is the inability to perform reverse genetic studies. Our objective is to demonstrate that by performing homologous recombination in cultured somatic cells and later using somatic cell nuclear transfer (SCNT) with those cells, fertile cloned offspring can be generated. A careful review of the data involving nuclear transfer experiments of all the species cloned to date revealed that small improvement has occurred since the original reports of SCNT (Thuan et al., 2010). The most important impediment to the development of new protocols is the lack of a suitable system that allows SCNT experiments on a large scale, at low cost, with a reasonably short endpoint, and readily- available reagents. Considering all these requirements, it is not surprising that mammals with long gestation periods or high per diem costs have not contributed to rapid advancement of the field. Zebrafish can be the ideal model to investigate factors that influence efficiency of nuclear transfer-cloning. B. Methods for transgenesis, forward, and reverse genetics in zebrafish Since the successful introduction of zebrafish as a model system, the number of transgenic zebrafish has grown exponentially in the past decades. According to the zebrafish information network (ZFIN), to date, there are thousands of transgenic zebrafish expressing fluorescence proteins (Sprague et al., 2008). Zebrafish embryos are transparent, enabling delivery of transgenes easily by direct injection. The approaches reviewed below are some of the most utilized by the research community. i. Transgenesis o Plasmid DNA injection and Meganuclease I-Scel Delivery of plasmid DNA to one-cell embryos has become a common approach to generating transgenic zebrafish (Niisslein-Volhard and Dahm, 2002). It consists of simply injecting the plasmid DNA into embryos and later identifying the transgenic animals. However the rate of transgenesis is low, and its expression is unpredictable. Transgenesis rate can be improved by using I-Scel meganuclease-mediated transgenesis (Grabher et al., 2004; Thermes et al., 2002). This is done by modification of the plasmid construct: having the transgene flanked by insulators and I-Scel recognition sequences (18 bp), and delivery by injection of the plasmid DNA together with l—Scel enzyme to one-cell embryos. It is still unclear how l-Scel improves transgenesis, yet it facilitates functional integration of the transgene and stable germline integration. 0 mRNA transfer for transient expression and caged mRNA The injection of mRNA to produce transient expression of transgene in embryos is well described (Nt'isslein-Volhard and Dahm, 2002). The transgene can be expressed instantly once delivered. The mRNA is commonly produced by in vitro transcription (the most widely used is Mmessage Mmachine SP6 kit, from Ambion), as capped mRNA. This transient effect of transgenes can be used for many purposes such as the study of transient effect of the transgene at a particular developmental stage, rescue of the phenotype of mutant embryos, delivery of functional genes, i.e., transposase enzyme for transposon-mediated mutagenesis and direct gene targeting by zinc finger nucleases, among others. The recent development known as photo—mediated gene activation using caged mRNA, allows for more control over time as well as site of gene activation following mRNA transfer (Ando et al., 2001). In this technique, the translation of caged RNA is inhibited by a photo—removable protecting group. Depending on the types of caging molecules, the caged RNA can be reactivated by a specific wavelength of UV light following a photolysis of caging molecules (K30, 2006). When the UV light is introduced at a specific location/tissue, mRNA is released for translation. Thus, it allows for studies of gain-of-function in a time and specific place. Note that the caged RNA is more stable than mRNA, and can be maintained up to 17 hours after RNA transfer, facilitating cell . tracing studies (Ando et al., 2001). ii. Random mutagenesis Forward genetics is routinely performed in zebrafish and consists of studying phenotypes prior to the identification of the responsible gene. This is facilitated, in part, by many of the desirable characteristics possessed by zebrafish, including its fecundity, external fertilization, rapid embryonic development and a short generation interval. Despite the lack of a reliable reverse-genetic approaches - those in which a mutation of a specific gene is induced and later its phenotype studied - forward genetics coupled with the aforementioned beneficial traits found in zebrafish, have promoted the study of large-scale mutagenesis in this organism, making zebrafish the first vertebrate used in these types of studies, and therefore, a great complement to D.melanogaster and Celegans models. Random mutagenesis approaches, while successful in generating mutant fish, usually calls for large-scale screenings that are laborious, costly and time consuming. In addition, not all mutants produced display a phenotype, as the mutagenesis often causes null phenotypes. 0 Random point mutation by ENU Germ line mutagenesis in zebrafish can be done by chemical means. N-Ethyl-N- Nitrosourea (ENU) is an alkylating agent that generates random mutations in the germline. This chemical mutagen is widely used in the zebrafish research community , (Grunwald and Streisinger, 1992; Solnica-Krezel et al., 1994). The mutagenesis process calls for consecutive treatment of adult-male zebrafish with ENU to generate random point mutations in spermatogonia. The treated fish, carrying mutated sperm, is then 10 bred to produce mutant offspring. The mutated genes can be later identified in the offspring by positional cloning, candidate gene approach, or target-selected screening as described below. Because founder fish contain mosaic mutations among their spermatogonia, thousands of mutant lines can be generated using this approach (Haffter et al., 1996). The sperm of treated male fish can be stored frozen in a library prior to the retrieval of affected gene(s) information following the phenotypic screening. Unfortunately the mutated genes are not easily identified, demanding extensive molecular screenings that are laborious and time consuming (Driever et al., 1996; Haffter et al., 1996). Most of the point mutations generated result in silent rather than missense or nonsense mutation, thus producing a null phenotype in F1 generations that cannot be recovered. Nonetheless, to date, more than 200 mutant lines with developmental phenotypes from ENU mutagenesis have been identified (Amsterdam and Hopkins, 2006). o lnsertional mutagenesis by pseudotyped retrovirus Retroviral-mediated insertional mutagenesis has been proposed and successfully launched by the Hopkins Lab (Lin et al., 1994). This approach utilizes the nature of retrovirus infection to assist mutagenesis, meaning that the RNA virus can convert itself to DNA (provirus) and randomly integrate its genome to an infected hosts’ DNA. The retrovirus is tropism, requiring compatibility of its envelop-proteins and the host receptor for infection. The pseudotyped retrovirus is genetically engineered to be replication-defective and to accommodate high infectivity in zebrafish cells. It utilizes viral genome and core proteins of Moloney murine leukemia virus, and the envelope 11 glyco—protein of a pantropic vesicular stomatitis virus. The pseudotyped retrovirus can be delivered to zebrafish embryos at the blastula stage, which then generates mosaic mutations in germ cells of each founder animal. The progeny of each founder animal can inherit many different insertional mutations. However, only 1/3 of the viral inserts result in gene disruption (Golling et al., 2002). In addition to its remarkable transgenesis rate, the provirus also provides a landmark for screening mutated genes in the host genome since the inserted genes can be recognized by a 5'- or 3’- random amplification of complementary DNA ends (RACE). To date, there are more than 300 mutants recovered from this mutagenesis approach (Amsterdam and Hopkins, 2006; Amsterdam et al., 2004). o Transposable element: gene trapping and gene breaking Recently, two transposon systems have been successfully introduced for mutagenesis in zebrafish. The two most popular are the medaka TolZ transposable element (Kawakami and Shima, 1999), and the Sleeping beauty (Davidson et al., 2003). Both systems vary in the origin of the transposable elements, yet their mechanism of action is based on the same principle. The transposable elements, characterized by flanked DNA with inverted repeats, are capable of moving from one locus to another in the presence of transposase. The insertional mutagenesis can then be introduced by delivery of transposase mRNA together with the transposable element through direct . injection to embryos. The trapped gene can also be identified by 5’- or 3’-RACE. Similar to most insertional mutagenesis, the random integration usually causes a null phenotype, as the inserts land mostly in the intronic sequences. Many attempts have 12 been made to increase the disruption of the gene, among those included 3’ or 5’ gene trapping and gene breaking system (Balciunas et al., 2004; Kawakami et al., 2004; Nagayoshi et al., 2008). When gene trapping is used as an aid, the 5’ gene trapping system (or promoter trap) is simply done by including the splice acceptor (SA) sequences at the 5’end of an insert cassette, a reporter gene such as green fluorescence protein (GFP) in the middle, and the polyadenylation (pA) signal at the 3’end of an insert cassette. This results in expression of the reporter gene under the endogenous promoter of the trapped gene. Hence the reporter gene only expresses if the transposable element lands in the region downstream of an active promoter. The trapped gene is commonly disrupted its gene expression as a reporter gene is followed by the pA signal. Mutants can be easily sorted by the expression of the reporter gene. However, only 1/3 of reporter loci are in the correct reading frame and able to express functional reporters. The 3' gene trapping system is done in a similar manner, by including an exogenous promoter that drives the reporter gene expression, and following with splice donor (SD) sequences. This system utilizes the pA signal of a trapped host-gene for transcriptional termination. The exogenous promoter provided upstream of a reporter gene ensures a correct reading frame and proper expression of the gene. However, targeting the 3’ end of a gene usually does not disrupt the function of trapped genes, . and commonly causes a null phenotype. In addition, the transcripts are known to be unstable, with a highly compromised reporter gene expression. 13 Gene breaking system combines a gene finding cassette and a mutagenic cassette (Sivasubbu et al., 2006). The gene breaking cassette contains an SA, pA signal, exogenous promoter, reporter gene, and $0 from 5’ to 3’, respectively. The S’end of the cassette, containing the SA and pA signal, is used to trap the promoter and to terminate the endogenous transcript. The 3’ end, containing independent expression of a reporter gene and SD, can ensure gene trapping. The mutants can be selected by expression of a reporter gene. Sivasubbu, et al. (2006) reported 53% insertional mutagenesis into functional genes using this system. As gene trapping and gene breaking technology contain a reporter gene, they do not require phenotype-driven insertional mutagenesis screening. iii. Gene-targeting mutagenesis The completion of the human genome project has accelerated study of functional genomics in humans, as well as other vertebrate systems. As the zebrafish genome sequencing project is ending, many zebrafish orthologs of human genes have been identified, increasing the demand for studies of functional genomics in this vertebrate model system. Reverse genetics approaches are possible in zebrafish, however, much improvement is necessary. The technologies reviewed below have been widely used in the zebrafish research community and are an attempt to efficiently implement the forward genetics approaches described. 0 "LUNG Targeting Induced Local Lesions In Genomes (TILLING) utilizes the existing mutant library of EN U-induced mutagenesis, as described above, to perform reverse 14 genetic studies (Wienholds et al., 2003b). This approach is called target-selected gene inactivation instead of gene-targeting. The study begins with the identification of desired mutated-genes, then searches the mutants from the ENU-mutant library, and later utilizes the desired mutant in the study. The point mutation in a desired gene can simply be identified by hybridization of DNA of the mutant with that of a wild-type animal. The heteroduplex DNA, if mismatched, can be recognized and digested by endonuclease, CE! I. Later, the mismatch can be diagnosed using high-resolution denaturing polyacrylamide gel electrophoresis. Alternatively, DNA sequencing of the region of interest can be used to identify the mutation. It is possible that the point mutation in desired genes may give a null phenotype. However, since the gene-targeting approach is not yet available in zebrafish, TILLING is known as the most utilized knock- out approach to date. A good example of a mutant identified by this approach is the dicerl mutant (Giraldez et al., 2005; Wienholds et al., 2003a). 0 RNA interference RNA interference (RNAi) technology has been utilized widely for in vitro cultured cells. The RNAi is designed to have its short RNA sequences complement the targeted transcript (antisense). Upon delivery to the cells, it binds specifically to targets, makes double stranded RNA, and subsequently causes destruction of transcripts or inhibition of translation. As a result, the specific expression of a given gene is temporarily knocked-down. This approach has been successfully implemented in studies of functional genomics in mammalian cells. Despite the successful application of RNAi- based technology to in vivo nematode model systems (Fire et al., 1998), in zebrafish, the 15 RNAi seems to cause non-specific destruction of RNA in viva (Gruber et al., 2005; Li et al., 2000; Zhao et al., 2001). - Morpholinos An alternative antisense technology has been successfully implemented to knock-down gene expression in zebrafish (Nasevicius and Ekker, 2000). Morpholinos (MD) are oligonucleotides that contain morpholine instead of riboside moiety, and phosphorodiamidate instead of phosphodiester linkages. This replacement stabilizes M05 and its function for up to 120 hours post-fertilization in vivo (Smart et al., 2004). The targeted gene knock-down by M0 is mediated by a steric blocking of translation followed by destruction of transcripts, hence it has less non-specific effect than small interference RNA. MO can be delivered to embryos or adult fish in a tissue specific manner. Since the knockout fish is not readily available, most of the functional genomic studies in zebrafish have utilized MO. The advanced technology, adopted from caged RNA, allows for a conditional knockdown of the desired gene (Shestopalov et al., 2007). 0 Target gene inactivation in ES cells and chimeras The study of reverse genetics using embryonic stem (ES) cells - chimera system has been extensively conducted in mice. Knockout mice are routinely generated using gene targeting homologous recombination in ES cells, followed by the production of germline chimeric founder animals. The ES cell-chimera technology has allowed for the disruption of thousands of genes in mice and provides a valuable tool for studying gene function. By definition, ES cells have three important characteristics: they are capable of renewing themselves indefinitely, differentiation into derivatives of the three germ 16 layers, and generating germ-line competent chimeras. In zebrafish, the derivation of ES cells has been successful (Ma et al., 2001). The reported cells meet the defined characteristics and are capable of contribution to the germ line of chimeric fish, albeit a decline in the rate of germ line contribution over ES cell passages in vitro was reported. Gene-targeting by homologous recombination in zebrafish ES cells was also achieved (Fan et al., 2006), and the mutant ES cells can be preserved as frozen cells for a long period of time. Despite substantial efforts to date, however, there are no reports of a knockout zebrafish produced by this approach. This is possibly due to (i) the rate of germ-line contribution of the ES cells being initially low, (ii) the ES cells have undergone too many passages in vitro for the positive/ negative selection, or (iii) the homologous recombination event is rare in the zebrafish ES cells. 0 Gene knock-outl-in followed by fish cloning using SCNT In those species in which ES cells are not a feasible approach to make knock- out/-in animals, somatic cell nuclear transfer (SCNT) can become a method of choice for germ line genetic modifications (Kuroiwa et al., 2002; Lai et al., 2002; McCreath et al., 2000; Richt et al., 2007). This approach calls for gene-targeting by homologous recombination in cultured cells, and subsequently, transferring the mutant nuclei into recipient eggs. As a result, cloned animals will be mutants, just as their donor cell. One drawback of this technique in vertebrates is its inefficiency that in turn negatively impacts the overall cost. The first cloned zebrafish produced by SCNT using long-term- cultured cells was reported in 2002 (Lee et al., 2002), opening the possibility to use cultured cells to perform gene targeting followed by nuclear transfer. Unfortunately, the 17 technique as described by Lee and collaborators did not work in our hands and those of others. More work was required to optimize SCNT in zebrafish. o Zinc-finger nucleases Zinc-finger nuclease (ZFN) technology has been introduced to increase the efficiency of homologous recombination in cultured cells (Perez et al., 2008; Santiago et al., 2008; Urnov et al., 2005). ZFN proteins are genetically engineered to have zinc-finger subunits for recognition of specific DNA sequences of the target locus, and an associated restriction endonuclease for generating double strand breaks (usually referred to as lesions) at the targeted DNA site (Kim et al., 1996; Smith et al., 2000). The activity of ZFN depends upon a formation of a heteroduplex of two endonuclease subunits. Zinc-finger proteins of each subunit are designed to bind to sequences flanking a target locus which accommodates interaction of its associated endonuclease subunits. Note that the number of zinc-finger proteins constructed correlates to the specificity of binding to the targeted locus. Only when two units of zinc-finger proteins bind to a correct locus, and are in close proximity to each other, will the endonuclease subunits form an active heteroduplex, and subsequently introduce a lesion to the target locus. The mutations introduced by ZFNs is target specific, and since ,in most cases, DNA repair following ZFN- induced double strand breaks is by non-homologous ends joining, a mutation is produced. The lesions can be either deletion or insertion, ultimately causing disruption of the target gene. Recently, the ZFN technology has been utilized in germ-line gene targeting in zebrafish (Doyon et al., 2008; Meng et al., 2008). It is done by mRNA transfer of two ZFN 18 subunits into one-cell embryos. Although, the mutant animal normally harbors mosaic lesions, its germ cells are usually affected with a single lesion. Indeed, its mutant progeny bears the same lesion. As of yet, the combination of ZFNs and DNA repairing by homologous recombination has not been reported in zebrafish. iv. Conclusion remarks for transgenesis and mutagenesis In summary, substantial effort has been put towards implementation of methods for transgenesis and mutagenesis in zebrafish, broadening the appeal of this animal as a reliable model of human disease and development. However, each one of the techniques described above has its own limitations and numerous factors should be taken into consideration when implementing them (Table 1). To date, random mutagenesis seems to be the best approach to produce mutant fish lines. Nonetheless, large-scale screening is very laborious and, more importantly, gene targeting is not possible. In order to truly understand gene function, gene targeting, i.e. knock-in or knock- out, is the method of choice. Its advantages have been clearly demonstrated in mouse studies. In zebrafish, morpholinos and ZFN are the only two techniques available that can be used to generate mutants using reverse genetics. Morpholinos can effectively down«regulate gene expression of the desired gene. However, it is transient and if the inactivation of gene expression is not complete, non-specific phenotypes can be observed. ZFNs have been shown to produce germ line competent mutants at reasonable mutagenesis rates, but it is still not amenable to knock-in experiments. 19 ES cells-chimera and SCNT are probably the most promising techniques for gene targeting in zebrafish. Of the two, SCNT is the best since it would require the least amount of time to generate a founder animal. At the moment though, the low rate of homologous recombination in cultured cells in both ES cells and somatic cells, hinders the broad applications of the technique. Problems associated with the low efficiency of germline transmission of ES cells and poor developmental rates in SCNT embryos are an additional drawback. It is possible that by combining some of the techniques reviewed, the ultimate goal of creating a knock-in/ ~out zebrafish can be achieved. As previously reported, ZFNs can increase the rate of DNA repair mechanisms, and may be used to generate cultured cells with gene targeting homologous recombination. 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The cytoplasm of oocytes, not the nucleus, contains maternal factors that potentially implicate the development of cloned embryos. While SCNT protocols may vary among species, the stage in the cell cycle in which the egg is best suited for cloning remains mostly constant, i.e., arrested metaphase Il (MII). This review focuses on the physiology of zebrafish eggs from the time of maturation to activation. i. The oocytes/eggs for nuclear transfer The first successful nuclear transfer experiments were conducted with amphibian eggs of Rana pipiens and Xenopus laevis (Briggs and King, 1952; Gurdon et al., 1958; Gurdon and Uehlinger, 1966). In part, this was due to their size since frog eggs (approximately 1 mm in diameter) are substantially larger when compared to mammals (approximately 100 pm in diameter), permitting microamanipulation by nuclear transfer without much demand for sophisticated laboratory equipment. Furthermore, matured frog eggs can be obtained in the thousands following hormonal injection. The Xenopus eggs are collected in high salt medium, Holtfreter’s solution, and remain arrested at Mll until nuclear transfer. In Rana pipiens, prior to enucleation, the recipient eggs are subjected to activation to enter metaphase II enabling the visualization of the egg’s nucleus. Removal of the chromosomes from the egg can be done using the now ‘conventional’ mechanical method using a glass needle (Briggs and King, 1952) or by UV 22 irradiation (Gurdon and Uehlinger, 1966). Donor cells are prepared by gently disrupting the cell membrane with an injection needle or pre-treated with membrane permeabilizing agents (lysolecithin or streptolysin 0) prior to nuclear transfer (Chan and Gurdon, 1996). In Xenopus, simple pricking of the eg with an injection needle while transferring a nucleus can cause the egg to activate and start embryogenesis. In mammalian cloning, the recipient oocytes are mostly arrested at M II, obtained from either in vitro or in viva maturation. For in viva maturation, MII oocytes are collected from animals that were previously hormonally stimulated to produce large quantities of eggs, a process called superovulation. In farm animals in which ovaries can be easily accessible from the abattoir, oocyte in vitro maturation is the method of choice. In this case, oocytes at germinal vesicle stage are collected, and cultured under conditions that are optimal for obtaining the Mll oocytes. MII oocytes are usually enucleated using a glass pipette to remove the metaphase plate. Delivery of the donor nucleus can be done by simple injection (Wakayama et al., 1998) or by cell fusion (Willadsen, 1986). During mammalian cloning, oocyte activation is done by various techniques including an electrical pulse, ethanol, strontium, calcium ionophore as the first step, and in some instances, with the addition of an inhibitor of protein synthesis or protein kinase as the second step (Cibelli et al., 1998; Kato et al., 1998; Wakayama et al., 1998; Wilmut et al., 1997). The first successful nuclear transfer in Medaka fish, used cells from the blastula stage (Wakamatsu et al., 2001). The technique involved the collection of MII eggs from the abdominal cavity of a female fish, and holding them at MII stage in a balanced salt 23 solution. MII eggs were then enucleated by X-ray irradiation prior to nuclear transfer. The cell membrane of the donor cell was then gently disrupted and transferred to the animal pole of the eggs, a process that also triggers egg activation. The description of this technique, i.e., irradiation of the egg first followed by nuclear transfer does not explain the subsequent work that the same group later did. We speculate that irradiation of the recipient egg was not as efficient as first probably thought (Wakamatsu et al., 2001). In a follow-up manuscript, medaka fish was cloned using non- enucleated eg as recipient and cells of embryonic or adult origin as donors (Bubenshchikova et al., 2005; Bubenshchikova et al., 2008; Bubenshchikova et al., 2007; Kaftanovskaya et al., 2007; Niwa et al., 1999). Note that using such technique, the cloned fish usually bears triploid chromosomes since the donor cell is diploid and the egg still contain its haploid-maternal chromosomes. Occasionally, diploid-fertile fish can be obtained by this technique as well (Wakamatsu, 2008). in addition, haploid ES cells of Medaka fish were recently generated and have produced cloned fish following nuclear transfer (Yi et al., 2009), albeit at low efficiency. In the future, knock-in / out Medaka fish could be generated if the non-enucleated unfertilized eggs are used together with haploid ES cells, since they can be amenable to genetic manipulations in culture. Nuclear transfer in zebrafish was successfully reported in 2002 (Lee et al., 2002). MII eggs were collected using the stripping technique, by gently applying pressure to the abdominal cavity, and kept in Holfreter’s solution at the time of egg collection. Egg enucleation was done by removal of the portion of the egg’ 5 cytoplasm underneath the 24 polar body using a Hank’s balance salt solution with bovine serum albumin. Nuclear donor cells were gently disrupted in the injection needle prior to transfer to the egg. Although the authors described the use of MII stage eggs with the technique as described, it is possible that eggs had exited Mll stage. This is a conclusion we arrived at our laboratory after attempting to replicate the results. We have speculated that the collection media or the pronase used for dechorination (removal of a zona pellucida-like shell called chorion) triggered premature activation and therefore SCNT was not successful in our hands. In summary, the recipient cells for nuclear transfer in frog and fish can be either activated eggs or mature arrested eggs (Table 2). In the latter case, the reconstructed eggs are activated right at the time of nuclear transfer. The success rate to produce clones, however, is different across species (Di Berardino, 2006). In mammalian cloning, especially in mice, the recipient cells at the metaphase stage are thought to be the most efficient cells to support the development of cloned animals (Egli et al., 2007; Wakayama et al., 2000). We have found that the quality of the eggs, as well as the cell cycle stage in which the eggs are found at the time of nuclear transfer, is a determining factor governing the success of the procedure. A thorough understanding of the physiology of the zebrafish eg at the time of activation and/or fertilization is necessary. 25 Table 2 Summary of nuclear transfer in frogs and fish 00W at Enudeatlon Nuclear transfer Cloned animals from cells of Species time of NT Stalnnlng by by Activation Blastula Embryos Adult Ref Rana pipiens MI and MII no glass needle direct injection pricking Yes n/a Tadpole 1 Xenopus laevis MII no UV irradiation direct injection pricking Yes Yes Tadpole 2 X-rays . . . . . . Medaka MII no , _ . direct injection pricking Yes n/a n/a 3 irradiation diploidize . . . . . . Medaka no not remove direct injection pricking Yes Yes Yes 4 egg ln/al I - Zebrafish te o no blind removal direct injection Spontaneous n/a Yes n/a 5 prophase before NT . , inject through a Spontaneous abnormal f MII H33342 l f Y Zebra ISh aser "mg micropyle after NT (15m) n/a es embryos - n/a is not applicable - References (REF) 1 (Briggs and King, 1952; DiBerardino and Hoffner, 1983) 2 (Gurdon et al., 1958; Gurdon et al., 1975; Gurdon and Uehlinger, 1966) 3 (Wakamatsu et al., 2001) 4 (Bubenshchikova et al., 2005; Bubenshchikova et al., 2008; Bubenshchikova et al., 2007; Kaftanovskaya et al., 2007; Niwa et al., 1999) 5 (Lee et al., 2002) 6 (Siripattarapravat et al., 2009b) 26 ii. The zebrafish egg After stimulation by gonadotropic hormone (GTH), the oogonium enters meiosis and starts its development. Oocyte growth in fish at pre—vitellogenesis is mediated by GTH-l (follicle stimulating hormone). GTH-Il (Iutinizing hormone) is responsible for maturation of fully—grown oocyte: germinal vesicle break down, stimulating follicle cells to synthesize maturation inducing hormone (MIH), and inducing ovulation. MIH stimulates synthesis of the maturation promoting factor (MPF), and maintains it at a high level in the mature Mil-arrested oocytes. In zebrafish, the ovulation is completed after the female is exposed to a male fish at dawn. It is not clear what really induces the ovulation in zebrafish upon mating, but it is thought to be pheromones. In Medaka, hydrolytic enzymes are responsible for follicular rupture prior to ovulation (Ogiwara et al., 2005). The mature egg detaches from the ovaries as denuded eggs, and is held in the ovarian cavity until the female is ready for mating. The eggs are released upon the natural mating behavior, at the same time that the milt is released from the male fish. The gametes are then fertilized externally within a few minutes. The mating zebrafish release eggs and milt many times over the course of a single mating. In zebrafish, eggs at the mature arrested stage seem to be the most accessible samples. The female fish usually holds hundreds of mature eggs in the ovarian cavity. These eggs can be released from the abdominal cavity of the female following the natural courtship behavior of the mating pair (Westerfield, 1993). Alternatively, germinal vesicle oocytes from the fish ovary can be obtained using a combination of the 27 enzymes trypsin, collagenase and hyalurodinase (Guan et al., 2008). A reliable in vitro maturation technique has been reported by using Leibovitz L~15 medium supplemented with 17alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and bovine serum albumin at high pH (Seki et al., 2008). The quality of the in vitro matured eggs is comparable to one of the in viva counterpart. However, since the mature arrested eggs are easily prepared, the in vitro maturation system is not yet implemented for the preparation of recipient cells for nuclear transfer. Scanning electron microscopy studies revealed that the sperm of zebrafish must enter the eg through a micropyle, a single cone shaped entrance on the chorion (Wolenski and Hart, 1987). At the time of exposure to water, the fish egg undergoes egg activation independent of the contact with sperm. The sperm must find the micropyle within seconds of ovulation or fertilization fails. The process is facilitated by the abundance of sperm surrounding the eg; the micropylar grooves surrounding the micropylar pit (Amanze and Iyengar, 1990); and the chorionic glycoproteins that can promote binding affinity of the sperm to the micropyle area as well (lwamatsu et al., 1997). Only one sperm per egg can go though the micropyle (Wolenski and Hart, 1987), and when one does, a filamentous actin network in the egg helps the fusion of the sperm to the egg at the fertilization cone (Hart et al., 1992; Wolenski and Hart, 1988). The major mechanism of egg activation is similar across all species. The activated egg increases intracellular calcium concentration in waves that vary in frequency and magnitude these are known as calcium oscillations (Ducibella and Fissore, 2008). In zebrafish, the egg undergoes parthenogenetic activation as soon as it comes into 28 contact with water, and a single calcium wave is recorded (Lee et al., 1999). The activated eggs can also show other signs including exocytosis of cortical granules, metaphase exit to complete meiosis, extrusion of the second polar body, expansion of the chorion, and the formation of blastodisc after the ooplasmic segregation toward the animal pole of the egg. The artificial oocyte activation methods used in nuclear transfer of mammals is not as efficient as fertilization, because only the single-exponential calcium wave is achieved after treatment. In bovine and murine, phospholipase C—zeta cRNA mimicks the calcium oscillation pattern of fertilized oocytes, and can promote nuclear reprogramming (Ross et al., 2009). In zebrafish, it is thought that egg activation by fertilization is not much different from the one of parthenogenesis since both produce a monotonic wave following activation (Lee et al., 1999). New line of evidence suggests that fertilization, not parthenogenesis, activates a Src-family protein kinase, the Fyn kinase (Sharma and Kinsey, 2006; Wu and Kinsey, 2000). The same group also showed that calcium oscillation are separated in two compartments, one that occurs at the center of the cytoplasm, and another one that starts from the micropyle and diffuses cortically throughout the cortex of the egg (Sharma and Kinsey, 2008). The latter is shown to be a sperm-specific wave and is different from the one triggered by parthenogenetic activation. 29 CHAPTER 2 THE DONOR CELLS FOR ZEBRAFISH SOMATIC CELL NUCLEAR TRANSFER: OPTIMIZATION OF IN VITRO CULTURE CONDITIONS, DNA TRANSFECI’ION, AND CHEMICALLY-ASSITED EPIGENETIC MODIFICATIONS. Kannika Siripattarapravat, Chia-Cheng Chang, and lose B Cibelli Abstract We characterized the essential parameters of donor cells for somatic cell nuclear transfer including culture conditions, growth and characteristic of cultured cells, and transfection efficiency of cultured cells. We also investigated the possibility of using histone deacetylase inhibitors, Trichostatin A (TSA) and Valproic acid (VPA), to treat the donor cells prior to somatic cell nuclear transfer. Introduction In somatic cell nuclear transfer (SCNT), there are at least three main factors that play a role in the success or failure of the procedure: the SCNT technique, the oocytes, and the donor cells. In simple terms, the reprogramming unit — referred to as the oocyte’s cytosol, must be exposed to the appropriate substrate — referred to the donor cells’ nuclei — and given substantial amount of time to completely remodel the somatic cell’s epigenetic state to a state of pluripotency. It has been suggested that modifying the chromatin structure of the somatic cells prior to their exposure to the egg’ 5 cytosol, may enhance the ability of these cells to be more readily reprogrammed following transfer into recipient eggs (Loi et al., 2003- l Simonsson and Gurdon, 2004) creating a greater capacity for normal embryonic 30 genes can be reactivated, and subsequently trigger an array of both, morphological and physiological changes in cells. Enzymes that modulate the epigenetic status of cells were found to be conserved among plants, invertebrates, lower vertebrates and mammals. Xenopus egg extracts can reprogram both mouse and human somatic cells to express a pluripotent marker, the OCT4 gene, which is regulated by DNA methylation (Byrne et al., 2003; Simonsson and Gurdon, 2004). The inter-Species cross reactivity implied that functional properties of these enzymes are also conserved. There is evidence of epigenetic modification enzymes in zebrafish which suggest their roles as gene expression regulators as well (Mhanni and McGowan, 2004; Yokomine et al., 2006). Multiple enzymes work in concert to equilibrate levels of DNA methylation and histone modifications in cells (Lachner et al., 2003; Shi and Whetstine, 2007). These enzymes are known to interact with different associated-regulatory proteins and are thought to recruit and work at preferential sites on the chromatin. Each enzyme also has its counter-partners that balance and maintain their substrates for regulation of gene exDression (Freitag and Selker, 2005). Many reagents were investigated to globally alter 31 the epigenetic status of the cells, including histone deacetylase and DNA methyltransferase inhibitors (Baurc'his et al., 2001; Kishigami et al., 2006; Rybouchkin et al., 2006; Sullivan et al., 2004). However, the effects of these reagents are known to be non—specific, inconsistent from cell to cell, dependent upon the epigenetic status of each cell at the time of treatment. In all cases, the inhibitors are thought to modify global epigenetic status of the cells by loosening the chromatin structure and favoring accessibility of transcription factors to the promoters. Histone deacetylase inhibitors, i.e., TSA, ultimately induce histone hyperacetylation as previously stated. Histone methyltransferase inhibitors decrease overall histone methylation, and DNA methyltransferase inhibitors, i.e., 5-Aza- 2’-deoxycytidine , help open/ loosen the heterocentromeric regions in the chromosomes making the chromatin easily accessible for the transcription factors to promote gene expression. Valproic acid, a drug widely used therapeutically in humans, has been found to trigger replication-dependent and -independent DNA demethylation through its effects on histone acetylation (Detich et al., 2003). In zebrafish, embryogenesis happens very rapidly following fertilization (Kane and Kimmel, 1993; Kane et al., 1992). This would require, in the case of SCNT, that the donor cell must be fully reprogrammed within 3 hours to reach the time of zygotic gene activation with the proper epigenetic modifications. We hypothesized that epigenetic alteration of donor nuclei prior to nuclear transfer, can potentially implicate the success of nuclear transfer and cloning in zebrafish. Therefore, the epigenetic state of donor 32 cells must be characterized. This information should be beneficial to increase the reprogramming efficiency through epigenetic modifications of the donor cells. Established primary zebrafish cell lines are in short supply. The in vitro system for culturing embryonic stem cells in zebrafish (Fan et al., 20043) and primary cells derived from embryos and adult tissues (Collodi et al., 1992; Driever and Rangini, 1993; Ghosh and Collodi, 1994) was previously described. However, there is limited information on the specific nature of the cultured cells and their optimal in vitro culture requirements, especially while using 5% CO; with atmospheric air. The types of cells and ability to genetically manipulate these cells are also left to be explored. Our objectives were then to first optimize cell culture conditions and to characterize cultured zebrafish somatic cells from embryonic source. The ability of these cells to uptake the foreign DNA, using several transfection mediators, was also evaluated. As we obtained the optimal culture condition, we further aimed to characterize the donor cells following global epigenetic modifications by chemical means and render them more susceptible for reprogramming in the egg’ 5 cytosol under the window of time of 3 hours or less. We expect that the use of modified donor nuclei will then increase the SCNT efficiency in zebrafish. Bssutts. i. The culture media The cell culture conditions were optimized by comparing two types of medium, Dulbecco’s Modified Eagle Medium (DMEM) and Keratinocyte SFM (K), with different combinations of supplements including, fetal bovine serum (FBS), trout serum (T5), 33 zebrafish embryonic extracts (EE), N-AcetyI-L—Cysteine (NAC) and L-Ascorbic acid 2- Phosphate sesquimagnesium salt (AZP). In assorted culture conditions, cell growth in different cell seeding densities were also determined. 0 Effect of TS and EE supplemented in DMEM on cell growth Embryo-derived cells were plated in 5% or 10% FBS in DMEM, either supplemented with TS or/and EE, or without supplement (Figure 1). There were no significant differences in growth rate between 5% and 10% FBS supplemented DMEM. The effect of 0.1% EE was shown to be minimal. Only the presence of 1% TS in culture medium significantly increased the growth rate of zebrafish cells (p<0.05). 0 Growth of zebrafish cells depends upon cell seeding density Primary cultured cells were derived from zebrafish embryos, 15-25 hours post- fertilization (hpf). Two different cultured media were compared; 1) DMEMs; DMEM with 15% FBS, 1% T5, 0.1% EE, and 10 ng/ml Bovine insulin and 2) K-NACs; Keratinocyte SFM (comes with epidermal growth factor and bovine pituitary extracts) supplemented with 5%FBS, 1%TS, 2mM NAC and 1 uM A2P (Lin et al., 2005). During the first 2 weeks, 25 ng/ml of basic fibroblast growth factor (Invitrogen) was added to the medium to inhibit melanocyte formation (Bradford et al., 1994). Different cell-lines and passages were then compared. Cells in low seeding concentration; 5 X 104 (p=0.003) and 1 X 105 (p=0.007) grew significantly better in K-NACs than DMEMs (Figure 2). No difference was found at 2-3 X 105 cell seeding density (p<0.05). However, at 4-5 X 10'5 cell seeding density, cells grew slightly better in DMEMs than K-NACs (p<0.1). We observed that cells plated more 34 efficiently in K-NACs than in DMEMs. The results suggest that cells propagated better at a higher cell seeding density and that K-NACs may improve plating efficiency, likely due to the presence of NAC and A2P in the media. 0 The growth of zebrafish cells at different %FBS and cell seeding density We determined the effect of NAC and A2P on cell growth in DMEMs (D—NACs) at different FBS concentration (Figure 3). Independent of the cell seeding density on day 1 of culturing, no difference was found for cell growth in either media supplemented with 5%, 10% or 15% FBS (p<0.05). Cells cultured in D-NACs grew significantly better than those cultured in DMEMs (p<0.05). Our results clearly show that the use of NAC and A2P in the culture medium has a positive effect on cell growth regardless of the amount of FBS added. This result further supports that the presence of NAC and A2P can increase cell plating efficiency, and possibly enhance propagation of cells. 0 Growth of cells in D-NACs and K-NACs Experiments were carried out to compare the growth of cells at different cell seeding density in two media; D-NACs (DMEM based) and K-NACs (Keratinocyte SFM based), supplemented with 5% FBS, 1% TS, NAC and A2P. Only in D-NACs 10 ng/ml bovine insulin and 0.1%EE were added. There was no significant difference in the growth rate observed between these two culture media (Figure 4). These results also indicate that cell growth in two different media is density- dependent. The doubling time and cumulative population doubling level were calculated as described previously (Lin et al., 2005). Doubling time of embryo-derived cells was recorded over multiple passages in vitro, using embryo-derived cells from embryos at 15 3S or 25 hpf primarily cultured in either D-NACs or K-NACs media. The doubling time of early passage (pl-5) was at 5—6 days in D~NACs, and 6-7 days in K-NACs. In both types of media, cells were at passage 5-10 and cultured for more than 10 passages. Doubling time was at 5-6 days for cells at passage 5-10 and 2-3 days for cells over passage 10. Cumulative population doubling levels exceeded 50in some cell lines cultured in either medium. The activity of telomerase was also evaluated in some of these lines at passages 2 and 20 (Figure 5), using TRAP assay (Chemicon, MA). The results showed that zebrafish cells, long-term cultured in either D—NACs or K—NACs, still have high telomerase activity, at the same level as earlier passaged cells. It remains to be determined if the high cumulative population doublings and telomerase activity detected in our Zebrafish primary cell cultures is cell type or cell culture dependent, i.e., if we have randomly selected tissue specific stem cells or our culture conditions were responsible for the seemingly robust in vitro cultures. 0 Characterization of embryooderived cells The types of cells that present in the culture were characterized by immunocytochemistry. We investigated for the expression of Vimentin, Sox17, Nestin and Oct-4, these are markers for fibroblasts, endoderm, neuronal progenitors, and pluripotent cells, respectively. When the analysis was done at early passages, the population of cells was heterogeneous (data not shown). In late passages however, the majority of cells in both D-NACs and K-NACs media were positive for Vimentin (Figure 6). None of them were positive for 50x17, Nestin or Oct-4 markers (data not shown), indicating that our cells were most likely fibroblasts. 36 ii. Transfection efficiency We aimed to define the parameters necessary for an efficient introduction of foreign genes into cultured cells prior to SCNT. We focused on somatic cells derived from zebrafish embryos and transfected them with foreign DNA. The expression plasmid vector containing Medaka elongation factor 1-alpha promoter driving green fluorescence protein (pEFla-A-GFP) expression (Kinoshita et al., 2000) was used. These cells were transfected using four different liposome-mediated transfection reagents: Lipofectin (Invitrogen, CA), Lipofectamine2000 (Invitrogen, CA), Fugene6 (Roche, IN) and Exgen500 (Fermentus, MP). DNA uptake rate, evaluated by transient transfection, was quantified by counting the percentage of green cells out of the total number of cells in culture (Figure 7a). Transfection efficiency was invariably low across all reagents tested, except Exgen500 which yielded the highest transient transfection efficiency in its group (p<0.05), although large variations were found within replicates. Electroporation was introduced as an alternative to Iiposome-mediated transfection reagents. We tested several combinations of Amaxa Nucleofactor® programs and solution kits as per the manufacturer recommendation (Lonza, Switzerland). The combination of program T-020 with solution kit V was used, as it gave highest transfection rate and best cell recovery. We used a pCSZ-GFP plasmid containing cytomegalovirus promoter driven GFP expression and found that more than 50% of the cells were found to transiently express GFP (Figure 7b). The transfection efficiency of electroporation is by far better than one of liposome-mediated agents. iii. Epigenetic modifications 37 We first determined the toxicity of TSA and VPA, which are both small molecules known to modify histone residues in the cells. We aimed to define an effective concentration for these inhibitors that is non—toxic to zebrafish embryos as well as capable of producing the desired modifications in the global histone acetylation status. Dose ranges were selected based on previous reports (Callas et al., 1999; Gurvich et al., 2005; Phiel et al., 2001). o Toxicity of TSA and VPA in zebrafish embryos Embryos were in vitro fertilized and place in a solution of either TSA or VPA within 5 minutes post-fertilization. Embryos were treated for 3.5 hours, then washed and moved to new egg water. We tested the TSA at concentrations of 5 nM, 50 nM, 100 nM, 500 nM, 5 nM, and 10 nM, and the VPA at concentrations of 0.1 mM, 0.5 mM, 1 mM, and 5 mM. Signs of embryonic toxicity for TSA were found at the 500 nM concentration. Treated embryos stopped their development at gastrulation, although no detectable abnormality prior to this stage was observed. A dose higher than 500 nM of TSA was lethal for 100% of the embryos. No detectable abnormality in embryos at day 4 post fertilization was observed when 100 nM TSA was used. The VPA caused abnormality of embryos after segmentation period when a concentration of 500 uM or more was used (Figure 8). The embryos showed signs of retarded growth, blunt tail, and pericardial edema. All treated embryos died at day 4 post fertilization. There was no detrimental effect when a concentration of 100 (1M was used. Therefore, the concentration of TSA and VPA that were used in the following experiment was based on the upper limit of toxicity recorded in zebrafish embryos. 38 0 Effect of TSA and VPA on histone acetylation Both, TSA and VPA are known to be histone deacetylase (HDAC) inhibitors. So, the levels of acetylation of histone H4 at lysine 5 residue (H4KS) were analyzed in culture cells following the treatment. Cells were incubated in vehicle controls, TSA at 100 nM and 200 nM, or VPA at 100 nM, 200 (AM and 500 nM, for 4 hours. Then cells were fixed and analyzed for acetylation at H4K5 by immunocytochemistry. The treatment with 100 nM TSA was sufficient to inhibit HDAC and cells displayed a marked increased in the levels of histone acetylation after treatment (Figure 9). In contrast, the treatment of VPA at 100 (M had no effect on histone acetylation (data not shown). We then increased the concentration of VPA to either 200 uM or 500 nM, however, only a minimal effect on the level of acetylation at H4K5 was observed after 4 hours incubation (Figure 9). When incubation time was prolonged to 12 hours using 500 [AM of VPA, we observed more cells with an increased level of acetylation at H4K5 (data not shown). We did not find any sign of cell dead following both treatments in cultured cells. Discusgion We have established the optimal in vitro culture system for zebrafish cells, including the media and cell seeding density for better cell propagation. The approach to yield high transfection efficiency in the cultured cells was also described. We reported the toxicity of TSA and VPA in the embryos and the efficacy of both reagents to increase the levels of histone acetylation at H4K5. 39 Our results showed that trout serum stimulated growth of zebrafish cells when heat inactivation was applied prior to its use. Embryonic extracts also have a positive effect on the growth of cells as well, however special care must be taken to avoid contamination and the quality fluctuations between embryo batches prompted us to exclude it from the final media preparations. Cultured cells had better plating efficiency when NAC and MP were added to the media. Both NAC and A2P are known for their capacity to reduce oxidative stress of the cells, and possibly help maintain the culture conditions similar to those observed under a low oxygen environment (Lin et al., 2005). Zebrafish cells seem sensitive to trypsin- EDTA treatments used to dissociate the cells. The culture system we chose calls for the use of low calcium medium (LHC) instead of phosphate buffer saline to wash cells, as well as a low concentration of trypsin-EDTA for dissociation of the cells. It is possible that NAC and AZP may facilitate a quick recovery of the cells after enzymatic treatment or mechanical injury at the time of dissociation. As a result, more cells survived and propagated in the next passages. We clearly observed that zebrafish cells grow better at a higher cell plating density. In our experience, the cells rather go to quiescence when low numbers of cells are plated in the cultured vessel. We found that the doubling time of zebrafish cells is longer in early passages than later passages. We also found, that many cells died off at early passages, possibly because the culture was a mixed cell population and some of the cells were not capable of re—plating. It is possible that cells at early passages grew approximately at the same rate as those during later passages, however, from our calculations; the doubling time was extended as it was adjusted for the dead of those cells. Regarding cell survival and growth, there is no difference found between DMEM and K-SFM based media. In both cases, the culture conditions favored the growth and expansion of vimentin positive cells. The cells in either media, are capable of long-term in vitro propagation, as the cumulative population doubling level (50) is quite high, and the telomerase activity also remains high. In some of the embryo-derived lines, we observed chromosomal aneuploidy at their late passages (P15-20). This is consistent with other reports in embryonic cells of zebrafish, i.e., cells have genomic instability during prolonged culture (Driever and Rangini, 1993). Although we did not characterize all the lines established, we found that some of the embryo-derived cells indeed had a normal karyotype. We had used D-NACs medium to culture adult fin explants as well. The fin explants propagated well in D-NACs medium but their doubling time was long (34 days). Adult cells displayed normal karyotypes in D-NACs media, suggesting that the aneuploidy observed may be more related to the cell type rather than the culture conditions. The efficiency of DNA transfection using liposome-mediated reagents was low in all reagents tested, making it impractical for routine use. Alternatively, electroporation showed high DNA uptake in the cultured fibroblast cells and should be considered the method of choice when genetic modifications must be introduced in cultured cells. 41 Considering the concentration of TSA and VPA used, TSA was more toxic than VPA. However, it seems to be a more powerful HDAC inhibitor since 100 nM concentrations are sufficient to increase histone acetylation. In the case of VPA, although well tolerated at higher concentration, its efficacy as an inhibitor of HDAC remains low, at least in the cells tested. By increasing the length of time the cells are exposed to VPA, the level of acetylation at H4KS in some cells increased, suggesting either slow activity of the reagent or replication-dependent efficacy. It is also possible that VPA induces changes at other histone residues besides H4K5. Early developing embryos treated with VPA showed multiple signs of abnormalities after the exposure to the drug was discontinued. This indicates that treatment of VPA may affect gene expression of embryos in early development, triggering abnormal phenotypes later in their development. Much work remains to be done in order to elucidate the specific effect VPA has on zebrafish cells. Culture conditions and techniques have been established to optimize the in vitro culture system of zebrafish cells and to genetically manipulate the cultured cells. All of which can be beneficial to handlings of donor cells for somatic cell nuclear transfer. The study of chemical treatments of cultured cells has set the foundation for future experiments that will demonstrate whether these chemicals can increase the success rate of SCNT in zebrafish. mm; Primary cultures of embrywderived cells. Embryos were obtained from natural breeding of Tuebingen Zebrafish line, incubated in egg water at 28°C until 15-25 hour 42 post-fertilization. A pool of 50 embryos was utilized in each line. Embryos were de- chorinated in 3 mg/ml pronase (Sigma, MA) for 5 minutes, and moved to LHC basal media (Invitrogen, CA) with 100 ug/ml gentamicin. Embryos were disinfected in 0.04% sodium hypochlorite (bleach) for 3 minutes, washed extensively in LHC and followed by mechanical disassociation in LHC by pipetting. Cells were resuspended in specific culture media depending on the experiment, and cultured at 28°C with 5% C02 in atmospheric air. More than 3 different cell lines, obtained from different batches of embryos, were utilized for each analysis. To subculture the cells, they were washed twice with LHC and subjected to trypsinization using 0.025% trypsin-EDTA (0.05% trypsin-EDTA and LHC in a ratio of 1:1) at room temperature. As soon as the cells started to dislodge from the culture dish, 5% FBS in LHC was added to inhibit the activity of trypsin. Cells were counted, and plated at designated numbers in culture dishes. Culture media was replaced every 3—4 days and subculture was performed when cells reached 80-90% confluency. In vitro fertilization (IVF) and toxicity test. IVF was done as described in the standard protocol (Westerfield, 1993), with slight modifications. The eggs were briefly kept in Chinook salmon ovarian fluid at room temperature prior to IVF. The milts were collected from at least 3 males and kept in Hank’s balance salt solution on ice until used. The eggs and milt were mixed and activated using eg water then incubated undisturbed for 5 minutes. Subsequently, fertilized embryos were moved to new egg water with VPA or TSA at designated concentrations. Embryos were incubated with each treatment for 3.5 43 hours, then washed extensively with egg water and raised at 28°C. The development of embryos was recorded until 4 days post-fertilization. Transfection. DNA transfection was performed according to the manufacturer’ 5 recommendation. Plasmid DNA was prepared using a midi-prep as described in a standard protocol (Sambrook and Russell, 2001). Two pg of DNA was used in each transfection. Immunocytochemistry. Cells were washed in LHC twice and fixed with freshly prepared, cold, 4% paraformaldehyde for 5-7 minutes. The fixative reagent was then removed and phosphate buffered saline (PBS) was added. Cells were treated with 0.5% triton X100 in PBS and allowed 15 minutes for permeabilization before they were washed twice in PBS with 0.1% triton X100 (PBSTx). Five percent bovine serum albumin (BSA) in PBSTx was then used as a blocking reagent for 90 minutes. Primary antibodies were diluted using 3% BSA in PBSTx. Cells were incubated in designated primary antibody overnight at 4°C with gentle rocking. Cells were then washed with PBSTx, and incubated with the couterpartners — AlexaFluor" labeled secondary antibody (Invitrogen, CA) for 90 minutes. Cells were washed extensively with PBSTx and their DNA stained with Hoechst33342. Images were taken with CoolSNAPTM Pro camera using Image-Pro Express (Media Cybernetics, MD). We used the following antibodies in the experiments; primary mouse anti- vimentin antibody ( dilution 1:200, Sigma-Aldrich), primary goat anti-Sox17 antibody (dilution 1:200 , Santa Cruz Biotechnology), primary goat anti-Oct4 antibody (dilution 1:200, Santa Cruz Biotechnology), primary rabbit anti—Nestin antibody (dilution 1:200, 44 Abcam), primary rabbit anti-H4K5 acetylation antibody (dilution 1:200, Upstate), secondary donkey anti-mouse AlexaFlour S94 (dilution 1:500, lnvitrogen), secondary donkey anti-goat AlexaFlour S94 (dilution 1:500, lnvitrogen) and secondary donkey anti- rabbit AlexaFlour 488 (dilution 1:500, lnvitrogen). Statistical analysis. Each experiment was repeated at least 3 times. Data was analyzed with SigmaStat version 3.1 (landel Scientific, San Rafael, CA), using analysis of variance (ANOVA). The level of significance was set to a p-value of < 0.05. 45 Elm-£1 Effect of FBS, TS and EE on cell growth in DMEM. DMEM was supplemented with 10 ng/ml bovine insulin, 5% (solid) or 10% (blank) of FBS in combination with either 1% T5, 0.1% EE or both TS and EE. A total of 1X105cells/3.8cm2 were seeded. Total number of cells were counted at day 7 after plating. (Error bar = standard error) .5 W I I 5% FBS D 10% FBS N I Cell number per well (x105) none 1% TS 0.1%EE TS+EE Supplements added 46 Fjggrg 2 Comparison of cell growth in DMEMs and K-NACs. Cells seeded at 0.5, 1, 2, 3, 4 5 2 and 5 X 10 cells/ 3.8 cm were propagated in both media. Numbers of cells were counted at day 4 after plating. (Error bar = standard error) El DMEMS I K-NACS Cell number per well at day 4 (X105) 2 3 4 5 Cell seeding density (x10-") 47 Cell number per well at day 4 (x105) 14 12 10 + 5%FBS DMEMs —Fr— 10%FBS DMEMs + 15%FBS DMEMs +5%FBS D-NACs ~42; 10%FBS D-NACs + 15%FBS D-NACs 2 Cell seeding density (X105) 48 Fjggrg 4 Comparison of cell growth in D-NACs and K-NACs media. Cells were seeded at 5 2 at 0.5, 1, 2, 3, 4 and 5 X10 cells/ 3.8cm and propagated in both media. Numbers of cells were counted at day 5 after plating. (Error bar = standard error) 4 l D D-NACS I K-NACS Cell number per well at day 5 (X105) N 0.5 1 2 3 4 5 Cell seeding density (x10-") 49 Figgge § Telomerase activity by TRAP assay. The telomerase activity of cultured cells at passage 2 (a), passage 20 (c), and positive control cells (e). Control ladder (g) is from a positive control DNA of the TRAP assay. Lane b, d, and fare heat inactivated cells of a, c and e, respectively, showing negative results for TRAP assay. ..l' = lllllllll? -'~ Lo! 2...: h i... .. t: 5...: _\.OH I: a 4.4: I {I ll? 50 Figure 6 Expression of Vimentin in cultured cells. Cells were grown in either D-NACs (a,b) or K-NACs (c,d) and stained for Vimentin (green) at passage 1 (a,c) and passage 15 (b,d). Nuclear staining is shown in blue. Scale bar is 50 um. .- 51 Ejgu_rg_z DNA uptake rates of zebrafish cells using liposome-mediated transfection reagents (a), or electroporation (b). The image is merged from phase contrast and green fluorescence channel, as green color depicted expression of green fluorescence protein under pCSZ-GFP (b). Scale bar is 100 um. 0.8 A 0.6 0.4 0.2 o . uh fi ni- Lipofectin Lipofectamine Fugene6 Exgen 500 2000 Percent transfected cells 52 Figure 8 Toxicity of VPA in zebrafish embryos. Embryos at day 1 post fertilization, following the treatment of VPA at indicated concentrations. Treated embryos showed abnormalities at their segmentation period. Except control IVF embryos, all treated embryos died at day 4 post fertilization. Scale bar is 1 mm. ' ..tP'..-’ . '. '. . Control lVF ' ' VPA 1 mM VPA 5 mM 53 Figure 9 Level of histone acetylation in cultured cells. Acetylation of histone H4K5 in control cells (aeb, g—h), and treated cells with 100 nM TSA (c-d), 200 nM TSA (e-f), 200 uM VPA (i-j), and 500 uM VPA (k-l). Blue color depicts nucleus of the cells, and green color is levels of acetylation at histone H4KS. Scale bar is 100 um. 54 CHAPTER 3 CHARACTERIZATION AND IN VITRO CONTROL OF MPF ACIWITY IN ZEBRAFISH EGGS Kannika Siripattarapravat, Angela Busta, Juan Pedro Steibel, and Jose Cibelli Published in Zebrafish 2009, 6(1): 97-104 Mas! We describe the characterization of maturation-promoting factor (MPF) in zebrafish eggs and used different defined conditions to maintain its activity in vitro. MPF activity levels are high in freshly ovulated mature eggs and decline rapidly within 5 min after either fertilization or parthenogenetic activation. The MPF activity of eggs matured in vitro declines faster when the eggs are incubated in Hank's culture medium supplemented with 0.5% BSA (H-BSA) than when incubated in Chinook salmon ovarian fluid (CSOF). MPF activity in non-activated, aged eggs remains high in H—BSA supplemented with 75 pM M6132 or 10 mM caffeine, but neither M6132 nor caffeine can sustain high MPF activity in activated eggs. MGlBZ-treated eggs showed delayed completion of metaphase and extrusion of the second polar body. Nuclear staining of the activated eggs confirmed the correlation between their cell cycle stage and MPF activity at each time point. An embryo toxic effect was found when matured eggs were held in 100 uM of M6132 or 20 mM caffeine for 1 h. Calcium—depleted medium and 1,2— bis(o-aminophenoxy)ethane-N,N,N',N‘-tetraacetic acid also showed detrimental effects on the embryos. Conversely, nonactivated, aged matured eggs maintained high MPF activity and developmental potential when CSOF was used as a holding medium. 55 W Changes in maturation promoting factor (MPF) activity in matured zebrafish eggs through postfertilization zygotes have not been reported. This study primarily focuses on the characterization and in vitro modulation of MPF activity in zebrafish, with the long-term aim of defining the best recipient eggs to use in somatic cell nuclear transfer. In general, it is widely accepted that a more efficient protocol for nuclear transfer can be obtained when oocytes are at metaphase arrest at the moment of fusion with the somatic cell. A recent publication has shown that enucleated zygotes can be used as recipient cytosol, as long as they are arrested at metaphase (Egli et al., 2007). The data suggest that reprogramming factors are not depleted after oocyte activation but can be retained in the zygote (Egli et al., 2007). The first cloned zebrafish were also reportedly produced by transferring nuclei into unfertilized eggs (Lee et al., 2002). More work needs to be done, however, to determine exactly what stage of the zebrafish egg's cell cycle is most suitable for nuclear transfer. lmportantly, the efficiency of zebrafish cloning is very low. It could be possible to increase it by exerting better control of the egg's cell cycle. To accomplish this goal, it is necessary to first characterize and later attempt to modulate MPF activity. Mature zebrafish eggs arrest at the second metaphase of meiosis before spawning (Nagahama, 1994). Metaphase arrest is regulated by cytosolic factor, which inhibits the anaphase-promoting complex/cyclosome. Several pathways have been. shown to play roles in regulating activity of cytosolic factor and have been reviewed elsewhere (Schmidt et al., 2006). Briefly, regulation of activity of cytosolic factor is 56 mediated by (i) activity of MPF, (ii) the Mos-MAPK pathway, and (iii) the Erp1/Emi2 pathway. In Xenopus, these pathways have been recently described to be linked by p90rsk (lnoue et al., 2007; Nishiyama et al., 2007). Zebrafish MPF consists of a catalytic subunit, Cdc2, and a regulatory subunit, cyclin B (Yamashita, 1998). While the level of cyclin 8 changes in relation to the different cell cycle stages, Cdc2 in eggs is constitutively expressed and maintained at a constant level (Kondo et al., 1997). Therefore, the activity levels of MPF depend upon the levels of cyclin 8. Both, cyclin 8 levels and MPF activity are low in immature eggs, are high in arrested Mil eggs, and decline again once the egg is activated (Figure 10). Pretranscribed cyclin 8 mRNA aggregates in growing zebrafish eggs (Kondo et al., 2001). This stored/masked mRNA disperses in response to maturation-inducing hormone and is subsequently translated after additional polyadenylation (Kondo et al., 1997). Phosphorylation of Cdc2 at threonine 161 (T161) by Cdk7 forms active MPF and that promotes metaphase arrest. Cyclin Bin fish is degraded by the 265 proteasome. The truncated proteins are initially cut at lysine S7 and subjected to ubiquitination for further destruction (Tokumoto et al., 1997). Degradation of cyclin 8 results in reduction of MPF activity and promotes meiotic exit. Persistent cyclin B levels can be maintained by introduction of a nondegradable protein mutated at lysine 57 (Tokumoto et al., 1997), which prevents eggs from meiotic exit. Calcium signaling is important for egg activation at fertilization and for initiation of embryogenesis (Ducibella and Fissore, 2008; Webb and Miller, 2000; Webb and Miller, 2003; Whitaker, 2006). There is evidence of a role for calcium in promoting 57 meiotic exit. In Xenopus, degradation of Erp1/Emi2 is regulated by calcium/ calmodulin- dependent kinase II and Plx1 (Schmidt et al., 2005). During spontaneous activation of matured rat oocytes, cyclin Bl and Mos are degraded by a calcium-dependent proteasome pathway (Ito et al., 2007). No direct evidence demonstrates the role of calcium signaling in the meiotic exit of zebrafish eggs. However, intracellular calcium concentrations increase exponentially at the time of egg activation (Webb and Miller, 2003), and, as in other species, it is thought to trigger many downstream developmental pathways. Many reagents have been used to modulate MPF activity, including M6132, caffeine, and 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). M6132 is a potent proteasome inhibitor known to inhibit degradation of cyclin B. A broad range of M6132 concentrations have been used to block cell cycle progression from metaphase. In Xenopus eggs, 100 (AM of M6132 inhibits the degradation of cyclin B for up to 1 h (Chesnel et al., 2006). The effective, as well as reversible, dose of M6132 needed to block pig oocytes at metaphase is 10 mM for a period of 30 to 48 h (Chmelikova et al., 2004). In rats, 5 (M of M6132 maintains MPF activity for up to 105 min after oocyte collection. Rat oocytes treated with M6132 were reported to display higher MPF activity than controls, promoting premature chromatin condensation after nuclear transfer (Ito et al., 2005). Later reports showed that cyclin B and Mos levels were maintained in rat oocytes in the presence of 10 to 25 pM of M6132 (Ito et al., 2007). M6132 (5 (M) was shown to reversibly hold rat mature oocytes in metaphase up 58 to 3 h (Zhou et al., 2003). These data suggested broad efficacy of MG 132 across species; therefore, it should also maintain MPF activity in zebrafish eg as well. Caffeine is another potent reagent known to maintain MPF activity. It has been proposed that caffeine acts through stabilization of the MPF complex (Kikuchi et al., 2000). In the pig oocyte, 5 mM of caffeine elevated MPF activity and promoted metaphase arrest (Kikuchi et al., 2000). Caffeine, at a dose of 2.5 mM, maintained high levels of MPF during pig nuclear transfer; it subsequently promoted premature chromosome condensation and increased the number of reconstructed embryos (Kawahara et al., 2005). Caffeine increased MPF activity and MAPK activity in sheep oocytes at concentrations greater than 10 mM (Lee and Campbell, 2006). Further, incubation of Ioach fish embryos in 2.6 mM caffeine for 1 h was not found to be toxic (Kopeika et al., 2003). To date, there have been no reports describing the use of caffeine in zebrafish eggs. BAPTA is a potent calcium chelator. It chelates both intra-and extracellular calcium and is reversible. BAPTA could potentially be used to prevent calcium signaling and to block oocyte activation. At a concentration of 10 uM, BAPTA has been shown to prevent murine oocyte activation (Zernicka-Goetz et al., 1995). It produced a transient effect without affecting embryo viability. Porcine oocytes loaded with 10 uM BAPTA were also blocked from activation (Ruddock et al., 2001). Cyclin B and Mos levels were maintained in rat oocytes in the presence of 10 uM BAPTA with a significant decrease in polar body extrusion, a sign of release from metaphase arrest (Ito et al., 2007). The use of BAPTA in calcium-free medium could potentially inhibit calcium oscillation in 59 zebrafish eggs and, therefore, prolong the metaphase. All three reagents, M6132, caffeine, and BAPTA, can potentially be of great help in modulating MPF activity in zebrafish. The present study characterized MPF in zebrafish eggs before and after activation. In addition, the efficacy of reagents that potentially maintain MPF in the eggs was investigated, as well as their toxicity. The results will be applied to direct optimal methodology toward increasing success of cloned zebrafish production. 59.511115. 0 MPF activity, cyclin B, and phosphorylated Cdc2 in unfertilized and fertilized matured zebrafish eggs While MPF has been well characterized in some fish species (lwamatsu et al., 1999; Katsu et al., 1993; Yamashita, 1998; Yamashita et al., 1995), little information is available for zebrafish (Kondo et al., 2001; Kondo et al., 1997; Tokumoto et al., 1997). MPF activity was measured in matured zebrafish eggs upon activation either by fertilization or spontaneous parthenogenetic activation. The differential effect of these two different egg activation protocols on changes of MPF activity was also investigated. Similar to Medaka (lwamatsu et al., 1999), zebrafish MPF activity declined within a few minutes after egg activation (Figure 11A). No significant difference was found between fertilization and parthenogenetic activation (Figure 11A). Protein levels of cyclin B and phosphorylated Cdc2 (Cdc2p) declined in a similar fashion (Figure 118.). In addition, the declining pattern of MPF was correlated with mitotic changes in the nucleus of fertilized eggs (Figure 12). Nuclear staining revealed that anaphase ll of 60 meiosis occurred within 5 min postfertilization. Twenty-five minutes later, the zygote nucleus was condensed again and the first cleavage of embryo began at around 35 min postfertilization followed by the second cleavage 25 min later. a In vitro fertilization rate of arrested matured eggs aged in Chinook salmon ovarian fluid Zebrafish eggs undergo spontaneous activation at spawning, within seconds of contact with water. Morphologically, this can be observed as a detachment of the chorion and formation of the fertilization cone (Wolenski and Hart, 1987). Two major holding media are known to maintain the nonactivated stage of zebrafish eggs: Hank's balanced salt solution with 0.5% bovine serum albumin (H-BSA; osmolarity 290 mosmol/L) and Chinook salmon ovarian fluid (CSOF; osmolarity 298 [+ or -] 6 mosmol/L). H-BSA is reported to maintain the fertilization capacity of eggs for up to 1 h (Sakai et al., 1997), whereas CSOF has done so for up to 6 h (Corley-Smith et al., 1999). An experiment was conducted to reevaluate the capacity of CSOF to maintain eggs in vitro and the subsequent developmental potential of these fertilized embryos. As previously described (Carley-Smith et al., 1999) an egg's capacity to be fertilized is compromised when aged in vitro; the longer the period between collection and fertilization, the lower the developmental rate (Figure 13). Sakai, et 01. observed that the fertilization rate of eggs aged in H-BSA declined rapidly in 2 h, and no eggs were fertilized at S h (Sakai et al., 1997). The current study showed that, in contrast to H-BSA, CSOF could hold egg fertilization capacity for up to 6 h (Figure 13), although a significant decline of developmental rate was observed (p < 0.0001). A significant proportion of eggs held in 61 CSOF for as long as 6 h maintained their fertilization capacity and developmental potential to blastula stage (51%, p < 0.0001) and hatch stage (20%; p < 0.01). o MPF activity, cyclin B, and Cdc2p in arrested matured eggs aged in vitro To determine whether the decrease in fertilization capacity was due to a decline in MPF levels during in vitro incubation of arrested matured eggs, MPF activity was measured in eggs that were held in either H-BSA or CSOF. MPF levels declined in both holding media, although at significantly different rates. MPF level decreased faster in H- BSA than in CSOF (Figure 14). Within 1 h, the MPF level of eggs incubated in H-BSA dropped to 60% of its initial value, whereas the MPF level of eggs incubated in CSOF continued to maintain its constant value. The decline in the fertilization rates accompanied the decline in MPF activity. Eggs incubated in CSOF maintained the appearance of freshly isolated eggs (Figure 15). In contrast, eggs in H-BSA showed various degenerative changes, such as spontaneous activation, detachment of the chorion, and disappearance of yolk granules (Figure 15). The spontaneous activation observed in some eggs in the H-BSA group was accompanied by the degraded form of cyclin B protein found in a Western blot analysis (data not shown). The overall findings support the hypothesis that MPF activity directly correlates with fertilization rates in eggs incubated in H-BSA. o Toxicity of MG 132, caffeine, and BAPTA-acefoxymefhyl (AM) esfer in zebrafish embryos As described previously, MPF activity in mammalian oocytes can be maintained by M6132, caffeine, and BAPTA. To further evaluate the efficacy of these agents in 62 maintaining MPF levels in zebrafish eggs, 3 toxicity test was first conducted to determine what dosage and incubation time were not detrimental to embryonic development. Eggs were collected in CSOF and assigned to control and treatment groups. Either H-BSA or CSOF was designated as a control. The treatment groups included 1-100 uM of M6132 in either HBSA or CSOF for 1 h, 1-50 uM of caffeine in either H-BSA or CSOF for 1 h, and 5-10 uM of BAPTA-AM in calcium—depleted H-BSA for 0.5 to 1 h. After each treatment, eggs were extensively washed in H-BSA and immediately fertilized. Embryonic development at the blastula stage, 1 day and 4 days postfertilization, was recorded (data not shown). The first signs of M6132 toxicity, observed at a concentration of 100 pM, began on day 1 and continued until day 4 after fertilization. They manifested as an increase in the percentage of abnormal embryos. No toxicity was detected when lower concentrations (less than or equal to 75 (1M) were used for 1 h. Eggs treated with 75 (M of M6132 showed a normal appearance and were not different from eggs in H-BSA (Figure 15). While 100 uM of M6132 showed embryonic toxicity, nuclear staining of parthenogenetically activated eggs demonstrated that those eggs were capable of completing metaphase (Figure 16); however, the second polar body was not extruded as rapidly as in nontreated eggs. Caffeine produced significant detrimental effects on eggs at 50 mM. In embryos previously incubated for 1 h in 20 mM of caffeine or less, a slightly negative effect (on development was detected. However, when the treatment time was extended to more than 1 h, an increase in toxicity was observed, suggesting a cumulative detrimental 63 was observed in such eggs (Figure 16). Calcium-depleted H-BSA was used as a control for BAPTA-AM treatment. A detrimental effect on eggs was observed in the control group when incubated for 1 h. The addition of BAPTA-AM to the holding media produced a more detrimental effect than the controls. Concentrations as low as 5 pM of BAPTA-AM were toxic to the eggs after 30 min of incubation. BAPTA-AM was, therefore, excluded from further studies. 0 Effect of MG 132 and caffeine on MPF levels in matured eggs aged in vitro and in activated eggs The levels of MPF decline in zebrafish eggs aged in vitro and immediately after egg activation. This suggests that treatment of eggs with either M6132 or caffeine could be used to modulate MPF levels in these eggs. To minimize the spontaneous activation after egg collection, eggs were maintained in CSOF for approximately 1 h before starting the treatments. As in the toxicity tests, concentrations of 75 and 100 uM of M6132, and 10 and 20 mM of caffeine in H-BSA were used. In each drug treatment, including controls, eggs were aged in vitro by incubation at room temperature. MPF activity, that is, total cyclin B and Cdc2p proteins measured by a Western blot analysis, of in vitro- aged eggs remained high during the incubation period in CSOF, and in treatments with 75 MM M6132 in H-BSA, with 10mM caffeine in H-BSA, and in HBSA, respectively (Figure 17). 64 To investigate the effects of M6132 and caffeine on activated eggs, eggs were treated with either one of these two reagents for 1 h before activation. Egg activation was triggered by dechorination, treating them with 3 mg/mL pronase in the presence of either reagent, to simulate the protocol for somatic cell nuclear transfer. Note that eggs were dechorionated; otherwise, they could not be manipulated using a conventional nuclear transfer protocol (Lee et al., 2002). MPF activity declined within a few minutes in activated eggs, regardless of which tested compound was supplementing the holding media (data not shown). Nuclear staining revealed metaphase exit in activated eggs treated with 100 uM M6132 (Figure 16). Discussion This study characterized zebrafish MPF activity in both (i) activated eggs and (ii) mature arrested eggs aged in vitro. We found that while CSOF can maintain MPF activity in arrested mature eggs, M6132, caffeine, and BAPTA can be toxic for arrested mature eggs before fertilization. Attempts to modulate MPF activity in activated eggs using these reagents were unsuccessful. Only CSOF maintained high MPF activity in nonactivated, aged matured eggs. After in vitro fertilization, CSOF aged eggs were capable of sustaining normal development as well. These results also confirmed what others have observed in mammalian and in teleost systems (lwamatsu et al., 1999), that is, MPF levels in zebrafish also increase at metaphase and decline rapidly after the completion of metaphase in fertilized and in parthenogenetically activated eggs. The failure to observe a rapid oscillation of MPF activity during the first and second cleavages may have been due to technical limitations 65 (Figures 11 and 12), because the samples were analyzed in pools, and it is possible that eggs in the same pool were asynchronous with each other. Besides the rapid decline of MPF activity, cyclin 8 protein was shown to degrade when H-BSA was used as a holding medium. The decline in the rate of fertilization can be partially attributed to spontaneous activation, as activated eggs showed a detachment of the chorion and the disappearance of the micropyle, the region in the chorion where the sperm enters (Wolenski and Hart, 1987). Another explanation for this decline in the fertilization rate could be a continuous loss of egg viability and overall protein degradation in the holding medium used (Figure 15). Considering that a higher fertilization rate was achieved in nonactivated eggs than in activated eggs when intracytoplasmic sperm injection was performed (Poleo et al., 2001) it is reasonable to conclude that the egg activation process triggers not only morphological changes that hinder the fertilization capacity, but also irreversible physiological changes that underlie embryonic development. CSOF, H-BSA, M6132, and caffeine were tested in an effort to find a compound or set thereof that could maintain MPF activity and delay egg activation. The results showed that both M6132 and caffeine partly help sustain MPF activity in eggs aged in vitro. M6132 may have partly mimicked the effect of CSOF, because the level of cyclin B protein remained relatively high in treated eggs (Figure 17). However, there was no advantage in using M6132 rather than CSOF in terms of improving the developmental rate of in vitro fertilized-embryos and maintaining nonactivated egg quality (Figure 15). Even though caffeine-treated eggs might maintain high MPF, caffeine’s toxic effect, demonstrated by degenerative changes in appearance (Figure 15), chromatin 66 condensation (Figure 16), and embryo toxicity, makes it an unsuitable candidate for such use. While these agents seem to help maintain MPF activity in nonactivated eggs, none of them can alter MPF activity in previously activated eggs. Only delayed resumption of meiosis II was observed using M6132; however, an increase in their concentration only exacerbated its embryonic toxicity. The current results demonstrate that to maintain high MPF activity as well as fertilization capacity, none of the reagents tested can replace the fluid obtained from the ovaries of Chinook salmon. The Chinook salmon is a Pacific salmon that reproduces only once, at the end of its life cycle. Although the composition of CSOF has not been reported, several studies on the ovarian fluids of closely related salmonid species have been conducted (Coffman and Goetz, 1998; Lahnsteiner et al., 1995; Olsen et al., 2001; Rime et al., 2004). Some of the components of this fluid include heat-lacid-stable serine protease inhibitor, antibacterial substances (lectins), apolipoprotein A-l-1, and hormones. However, no direct evidence links any of these compounds with the ability to maintain the nonactivated stage of zebrafish eggs in vitro. There is speculation that the osmolarity of ovarian fluid may prevent egg activation in viva, though it is not the sole reason, as demonstrated by Sakai, et 0! using HBSA. Perhaps antiproteases act to prevent degradation of several proteins, including MPF, extending the egg viability period in vitro. The activity of MPF declines slowly over time in nonactivated eggs aged in vitro in CSOF, M6132, caffeine, and H-BSA, respectively. Toxic effects were found when 100 67 (1M M6132, 20 mM caffeine, and calcium-depleted medium were used as holding media before fertilization. The current study argues in favor of using CSOF to maintain nonactivated eggs for extended periods before performing somatic cell nuclear transfer or intracytoplasmic sperm injection experiments. Considering that activated eggs show irregular levels of MPF, the overall success of somatic cell nuclear transfer may fluctuate depending on whether the time of nuclear transfer matches the high point of MPF activity or not. Only nonactivated eggs with high MPF activity would be the best recipients for somatic cell nuclear transfer. Methods Egg/milt collections and in vitro fertilization. Eggs and milt from zebrafish (Tubingen strain) were obtained using the stripping technique described previously (Westerfield, 1993). Briefly, breeding pairs were set in individual tanks the day before the eggs and milt were collected. The male was introduced to a female tank in the morning after the light came on. Once breeding activity began, and before spawning, the female fish was immediately isolated from the male. Females were anesthetized using MS—222 (Sigma, St. Louis, MO), and the eggs were collected by gentle stripping and placed in either CSOF or H-BSA. CSOF was obtained from Chinook salmon (Oncorhynchus tshawytscha), kindly donated by the Michigan Department of Natural Resources. Eggs were selected under a stereomicroscope, and only those of good quality (yellowish and granular) were selected for subsequent experiments. At least three females were utilized for each experiment. 68 For milt collection, male fish were anesthetized in MS—222 and subsequently placed in the slit of a sponge with their tails hanging over and their genital openings at the edge. Excess water was dried out, and the milt was collected in a capillary tube under a stereomicroscope by gently stripping. Milt was immediately transferred to ice- cold Hank's solution. Before its use, a portion of milt was evaluated for sperm density and motility under a microscope. Good-quality sperm was of high density and nonmotile in Hank's solution, but became active swimming when egg water was added. Milt was collected from at least three males for each experiment, and each batch was used within 30 min of collection. Eggs were activated by either fertilization or parthenogenesis. We performed in vitro fertilization according to a core protocol from The Zebrafish Book (Westerfield, 1993). For delayed in vitro fertilization, either H—BSA (Sakai et al., 1997) or CSOF (Corley— Smith et al., 1999) was used as a holding medium. Parthenogenetically activated eggs were moved from the holding medium to egg water. To measure MPF activity, pools of 10 eggs or embryos were collected at designated times before and after fertilization or parthenogenetic activation. All samples were snap frozen in liquid nitrogen and stored at ~80°C. Cell lysates. Before use, samples were thawed and lysed in sample buffer (50 mM Tris, 0.5 M NaCl, 5 mM EDTA, 2 mM EGTA, 0.01% Brij35, 50 mM 2-mercaptoethanol, 25 mM [betaj-glycerophosphate, 1 mM Na-orthovanadate, and protease inhibitors), with a ratio of one egg per 2 (1L of sample buffer. 69 MPF activity by ELISA. MPF activity (p34Cdcz kinase) was quantitatively measured using a Mesacup Cdc2 Kinase Assay Kit (MBL International, Woburn, MA). The activity of p34”:dcz kinase measured by this kit showed high correlation coefficients (as high as 0.9961) to histone H1 kinase activity measured by a radioactive method (Ito et al., 2001). Results were read as the optical density (00) at 492 nm. The OD492 values of each sample were quantified as a percentage of sample MPF activity to control MPF activity at time zero. At least three biological replicates were performed for each experiment. Western blotting for cyclin B and Cdc2p. To confirm the presence of cyclin B and Cdc2p, the samples were analyzed by Western blotting using standard protocols (Sambrook and Russell, 2001). Briefly, lysates were prepared in SDS sample buffer, and according to its different molecular weight, cyclin B was analyzed in 10% SDS-polyacrylamide gel electrophoresis, while Cdc2p was analyzed in 12% SOS-polyacrylamide gel electrophoresis. Approximately 1 egg from the total pool lysates of 10 eggs was loaded to each lane. Proteins were separated by electrophoresis at 50 V for 4-6 h in Tris-glycine buffer and transferred onto the polwinylidene fluoride (PVDF) membrane at 4°C, 100mA for 4h in transfer buffer. Blots were blocked in 5% skim milk in Tris-buffered saline and 0.1% Tween-20 (TBST), with agitation for 90min at room temperature. Mixed mouse monoclonal lgGs primary anti-cyclin Bl antibody (Upstate Biotechnology, Lake Placid, NY) was diluted to 1 ug/mL in TBS. Goat primary anti-Cdc2p (T161) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was diluted to 1 ug/mL in TBST with 3% skim milk. Blots were incubated overnight at 4°C with each primary antibody and then washed extensively with TBST. Either horseradish peroxidase-conjugated goat anti- 70 mouse antibody or bovine anti-goat antibody (Santa Cruz Biotechnology) was diluted to 0.4 ug/mL in TBST with 3% skim milk. Blots were incubated with each counterpart secondary antibody at room temperature for 90 min and then washed extensively with TBST. Blots were immersed with SuperSignal West Chemiluminescent Substrate (Thermo Scientific Pierce, Rockford) for 5 min and subsequently exposed to X-ray films. Nuclear staining. Eggs were dechorionated in 3 mg/mL pronase and washed extensively in Holtfreter's solution. At the time of collection, samples were immersed in 4% cold paraformaldehyde for 12 to 24h. Fixed samples were washed in 0.1% Triton-X100 in PBS (PBSTx) for 20 min and then stained with 1 ug/mL Hoechst 33342 in PBSTx for 15 min. Samples were triple-washed with gentle agitation in PBSTx. Images were taken under a fluorescence microscope with Image-Pro Express (Media Cybernetics, Bethesda, MD). Statistical analysis. A two—way linear mixed model analysis of variance (Littell et al., 1996) was performed on the percentage of abnormal embryos. The fixed effects included in the model were treatment (16 levels), development stage (3 levels), and their interaction. The random effects of female and random interaction of female by treatment were included to account for repeated measures on the same individual. Model fit was assessed by analysis of the residuals. Our study revealed no evidence of association between treatment and development stage (p = 0.66). Significant main effects of treatment (p = 0.015) and developmental stage (p < 0.0001) were observed. Differences between the treatment levels were further assessed through pair-wise comparisons. Tukey‘s test was used to account for multiple tests, using an overall significance level of p = 0.05. 71 Figure 10 Morphological and molecular changes during parthenogenetic activation of zebrafish eggs. (A) Cdk7 phosphorylates Cdc2 at threonine 161 and when combined with cyclin B (ClnB) - present at high levels in mature eggs - form active MPF. (B) Mature eggs with high MPF activity are arrested at metaphase ll of meiosis. The micropyle is closely associated with the egg’ 5 chromosomes and the first polar body. (C) Egg activation triggers degradation of ClnB by ubiquitin-dependent proteasome pathway, decreasing the activity of MPF. (D) Activated eggs exit metaphase, extrude the second polar body, and form the female pronucleus. MPF == maturation promoting factor, Cdc2P = phosphorylated Cdc2, UB == ubiquitination, MII = metaphase ll of meiosis, lst PB = first polar body, 2nd P8 = second polar body, FPN = female pronucleus. EGG ACTIVATION High MPF Low MPF us i ue us Us 08 CdkT' 03 9 fig Cdc2P (3'nt . c Degraded ClnB I o Yolk , Chorion / )\ /2“d PB // F PN Arrested-mature egg 7 Activated egg 72 Figure 11 MPF activity in eggs activated either by fertilization or parthenogenesis. (A) Cdc2 kinase activity of activated eggs. (B) Western blot of cyclin Bl and Cdc2p of activated eggs. Time indicates minutes post-fertilization or minutes post- parthenogenetic activation. Error bars are standard error of means. A 120 ~ + F ertiization 100 + Parthenogenesis m o 1 Cdc2 kinase activity 0) o 40 '1 20 4 O I T r i i ‘1 0 10 20 30 40 50 60 Time (minutes) post-ferb‘lization or post-parthenogenetic activation B Time (minute) 0 1 2 3 4 5 1o 20 so 40 so Post Fertilization f CInB1 - . Cdc2-P II I IIIIA . _- cun31.------ an- P tPrth °‘ ‘ .....W“..... 73 Figure 12 Nuclear staining of fertilized eggs. Numbers indicate the time (min) after fertilization; metaphase (0 min/3 min), completion of metaphase (5 min), extrusion of second polar body (10 min), decondensed male and female pronuclei (25 min), beginning of the first cleavage (30 min), anaphase (35 min), 1 of 2 cells (40 min), decondensed nucleus (45 min), and beginning of second cleavage (55 min). Scale bar = 10 um. 74 Figure 13 In vitro fertilization rates of eggs aged in CSOF. Graph shows percentage of fertilized embryos obtained from eggs aged in CSOF for 1, 2, 3, 4, 5, and 6 hr post- collection. Natural breeding (NB) indicates rate of fertilization at time 0. Solid bars indicate blastula development, and open bars indicate hatched embryos. Error bars show standard error of means of three biological replicates. a? 80961 7096* 5096* 3096— Developmental rate of embryos 20963 10%~ NB (0h) 1h 2h 3h 4h Sh 6h Time of in vitro fertilization (hour post-collection) 75 Figure 14 MPF activity of in vitro aged eggs in either CSOF or H-BSA. (A) Cdc2 kinase activity. (B) Western blot analysis of Cdc2p. Time indicates hr post-collection. Error bars are standard error of means. 120 A 100 5‘ .5 80 u a o ‘3 6° ‘1' N - 8 40 . 0 2° +csor= —-—- HBSA 0 ' , ¥ 0 1 2 3 4 5 6 Time post-collection (hours) . I-I-BSA B Tme 1 2 3 (hours) __ g . . , 76 Figure 15 Pictures of arrested matured aged eggs in CSOF, H-BSA, 75 (1M M6132 in H- BSA, and 10 mM caffeine in H-BSA. Images were captured at the indicated hr post- collection (hpC). Images were taken from random fields. Identical results were observed from the three biological replicates performed. Only one of the three is shown here. Scale bar = 500 pm. Arrows indicate eggs with degenerative appearances at 3 hpC. m H-BSA 75uM M6132 10mM Caffeine 77 Figgre 16 Nuclear staining of parthenogenetic embryos. Eggs were incubated in 100IIM M6132, 20mM Caffeine and control medium (H-BSA) and then fixed at the indicated time of post-parthenogenetic activation. Following parthernogenetic activation, eggs underwent metaphase exit when either H-BSA or M6132 supplemented H-BSA were used, however, caffeine treated eggs showed chromatin condensation and no extrusion of second polar body. Scale bar = 10 pm. Time post-parthenogenetic activation Holding 3 min 5 min 10 min 20 min 30 min 40 min media H-BSA H-BSA , +100uM It . M6132 H-BSA +20mM Caffeine 78 Fi ure 17 MPF activity in matured eggs aging in vitro. Western blot analysis for cyclin BI and Cdc2p of mature eggs in H-BSA, CSOF, H-BSA with M6132 (75 or 100 uM), and H- BSA with caffeine (10 or 20 mM). Line numbers indicated hr post-collection. .... .... ---.-’::-e§o IIIIIIIIIIIIINé. o n v n N p a... m a, v. n u a n... :35 5.32.3 05.58 258 2.8.5 .282 38. «E: tltttittgi... nan-".....I. . .50 m u v n N _. m o m m w n a 9 ad carpooflwuo N30: .2103 ~30: .213. 32. 08:. .. .. ..- I j... ..-...3 .I .l I. 6"... ..l. ...... m a v n N r ad 0 w o. n N —. ad Chm—“Hun”; «69.: .2230 now“. .9580 62. 0E: 79 CHAPTER 4 SOMATIC CELL NUCLEAR TRANSFER IN ZEBRAFISH Kannika Siripattarapravat, Boonya Pinmee, Patrick] Venta, Chia-Cheng Chang, and lose B Cibelli Published in Nature Methods 2009, 6(10): 733-735 Received 16 June 2009; Accepted 29 July 2009; Published online 30 August 2009. Meet. We developed a method for somatic cell nuclear transfer in zebrafish using laser- ablated metaphase ll eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones. Introduction Zebrafish is a convenient, relatively inexpensive and useful vertebrate animal model for the study of normal and pathological development, physiology, aging and disease. Large-scale mutagenesis and screening have proven to work efficiently in this organism. However, these 'forward genetic' approaches are highly laborious and time- consuming (Anderson and Ingham, 2003). A simple 'reverse genetics' method is necessary to bring the zebrafish model system into parity with rodent model systems. Mutant knockout or knockin mice are routinely generated using gene targeting in) embryonic stem cells. Despite substantial effort (Fan et al., 2006; Ma et al., 2001), there 80 are no reports of transgenic zebrafish with germline transmission generated using this approach. Somatic cell nuclear transfer (SCNT) has the potential to become the method of choice for germline genetic modification in fish. A previous report of cloned zebrafish demonstrated that nuclear transfer with cultured cells is possible, with an efficiency of cloned fish production at 2% or less (Lee et al., 2002). However, the reproducibility of the protocol is poor. Multiple factors may be responsible for this, among them are the use of activated eggs as recipients, which limits manipulation time to less than 1 h after egg collection; the technical challenge of removing the egg's chromosomes by removing a portion of egg's cytoplasm underneath the second polar body without staining the DNA; and the manipulation of dechorionated eggs and handling of the fragile reconstructed embryos. We describe here a simplified methodology that addresses each of these problems. Our method relies on (i) the use of mature, arrested eggs at metaphase ll of meiosis as recipients, making use of the observation that mature eggs can be maintained in an inactivated state in Chinook salmon ovarian fluid (CSOF) for up to 6 h with negligible detrimental effects (Siripattarapravat et al., 2009a); (ii) complete laser inactivation of the Hoechst-stained DNA in the metaphase plate of the egg, leaving the egg's cytosol intact; (iii) delivery of donor cells through the micropyle, the route that the fish sperm uses to enter the eg, using a human intracytoplasmic sperm injectionneedle to transfer the nucleus into the animal pole of the egg; (iv) egg activation after nuclear transfer; and (v) manipulation of cloned embryos with intact chorion (Figure 18). Using 81 this approach, the egg remains at metaphase ll after reconstruction until activation in egg water (Figure 19a). Notably, in most of the species cloned to date, metaphase ll oocytes have shown to be the most suitable recipient cytosol for SCNT (Egli et al., 2007). In comparison to control parthenogenetically activated embryos (Figure 19b), embryos of the laser-ablated group showed an absence of female pronuclei and second polar body extrusion (Figure 19c). To facilitate phenotypic screening of clones, we used donor cells from two types of zebrafish: (i) golden fish (Lamason et al., 2005) in an AB strain background, in which homozygotes have golden phenotypes and heterozygotes appear as wild-type zebrafish, and (ii) zebrafish expressing GFP in the Tubingen long fin (TuLF) background (Nagayoshi et al., 2008). We isolated donor cells from the 15-20 somite-stage embryonic tailbud or cultured cells from adult caudal fin. We obtained recipient eggs from either wild-type zebrafish, transgenic zebrafish homozygous for histone HZA tagged with GFP (H2AzGFP) (Pauls et al., 2001) or a line derived by outcrossing Tubingen and AB lines (TAB). We monitored the golden phenotype or the green fluorescence of transgenic TuLF in clones produced by this technique. The use of golden donor cells in combination with wild-type recipient eggs produced golden fish (Figures 20a and 20b). The use of transgenic TuLF donor cells in combination with TAB recipient eggs produced GFP+ fish that showed a long—fin phenotype upon reaching adulthood. SCNT offspring using ++ recipient eggs from transgenic H2AzGFP fish provided evidence of complete inactivation of the egg genome, as they showed a loss of nuclear localized green fluorescence (Figure 20c). In addition, to confirm that no genetic material of the 82 recipient cell was carried over to clones, we performed single-nucleotide polymorphism (SNP) genotyping analysis (Table 3). The DNA fingerprint of cloned fish showed a complete match to that of a donor cell (Table 4). Approximately 40% of reconstructed embryos completed the blastula stage of development (Table 1). Of these, more than half paused between high (3 h) and sphere (4 h) stages, and later did not enter gastrulation. Clones that did not complete gastrulation (90% epiboly), usually completed the germ ring stage but did not form the embryonic shield, an involution of the hypoblast. Most of the clones from embryonic tailbud donor cells that completed 90% epiboly developed to 1 d (completed segmentation). For adult caudal fin donor cells, less than half of clones that completed gastrulation also completed segmentation. Using golden donor cells, approximately 2-15% of reconstructed embryos developed to 1-d-old fry (Table 5). Up to 2.2% of reconstructed embryos, using embryonic tailbud cells and wild-type eggs, grew to fertile adult fish (Table 5). Clones from golden fish reached reproductive maturity and when crossbred with a golden counterpart produced 100% golden offspring (Figure 20d). Offspring (F1 generation) of both golden clones were healthy and produced golden fish in the F2 generation. One of the adult golden fish died at 21 months (Figure 20b) and we killed the other at 16 months because of signs of emaciation. Using TuLF donor cells, 3.3—10.7% of reconstructed embryos developed to 1 d (Table 5). All of the clones expressed GFP. Six percent of reconstructed embryos using embryonic tailbud cells developed to eating fry and five clones survived to adulthood. At 83 the time of this writing, 3 clones were at 4 months of age, healthy and produced offspring carrying their genetic traits. We observed various extents of abnormality in 1-d—old clones (Figure 21). Most abnormal clones showed more posterior rather than anterior developmental defects, that is, all abnormal embryos showed primitive development of the head and eyes but defective tail formation. Most of the cloned embryos that did not develop to 4 d displayed severe abnormalities such as growth retardation, a bent tail, a small head, a lack of hematopoiesis and a short trunk. Clones that developed to 4 d but did not eat showed minimal abnormalities, including no swim bladder formation and enlarged pericardium. Despite any detectable abnormality, some clones died after 7—10 d, and some clones could eat but died at 12—20 d. In the latter cases, it is uncertain whether the death was caused by cloning or could be explained by common loss of fish embryos at an early age. In all cases, abnormal clones of adult caudal fin donor cells showed more progressive abnormalities than those of embryonic tailbud donor cells. We did not observe the 'no head' phenotype as reported by others (Lee et al., 2002). All clones examined had a normal karyotype (2n = 25) as shown by replication banding (Amores and Postlethwait, 1999)(Figure 22a). In addition, we used 11 SNPs to confirm the identity of cloned embryos, donor cells and female egg donors (Figure 22b). All cloned fish showed complete matched genotypes to one of the donor cells, but not to those of the female egg donors (Table 4). The use of metaphase ll eggs in CSOF allowed for longer manipulations sessions, approximately 50 eggs per person per day. For cell transfer, the use of intracytoplasmic sperm injection needles in combination with injection through the micropyle avoided premature egg activation from dechorionation by pronase (Siripattarapravat et al., 2009a). Additionally, eggs with an intact chorion were more tolerant to micromanipulation and injection, as naked eggs are easily broken by the suction of the egg holder or sharp-point injection needles. Our technique overcomes these difficulties, allowing us to manipulate eggs more practically and monitor developing cloned embryos independently. The combined use of SCNT and donor cells that can be grown in vitro would allow for the use of knockout and knockin methodologies in which the integration site and disposition of the transgene can be confirmed before generating cloned zebrafish. Furthermore, a permanent reservoir of cells with the desired genotype can be maintained in the form of cultured somatic cells and/or cryopreserved samples. Above all, the timeline to produce a founder fish carrying the targeted gene could be shortened by 6—7 months. A zebrafish SCNT procedure like the one described here could enhance the advantages of this model for studies of vertebrate developmental biology and human disease. Methods Zebrafish resources. For donor cells, we used homozygous golden (slc2405b1/ ”1 ) in the AB background (Lamason et al., 2005) and transgenic fish lines expressing GFP (H6n62A, HGn28A and HGnBE) in the TuLF background (Nagayoshi et al., 2008). Recipient eggs were obtained from wild-type, transgenic homozygous histone HZA-tagged with GFP 85 (H2AzGFP) in the AB background (Pauls et al., 2001) or outcrossed fish of Tubingen and AB line (TAB). Preparation of recipient eggs. Eggs were obtained from sound females using the stripping technique (Westerfield, 1993). Each female fish was sedated with M5222 and gently squeezed in the abdomen. Eggs were collected directly in CSOF and sorted for quality under a stereoscope. Eggs were immersed in 50 ug/ml of Hoechst 33342 in CSOF for 20 min and held in CSOF in a moist chamber at room temperature (23 °C) until used. Note that eggs exposed to both Hoechst DNA staining and UV-light irradiation protocols showed no detrimental effect of this treatment to embryonic devel0pment after in vitro fertilization (K.S., unpublished data). Just before manipulation, eggs were washed in 5% polyvinyl pyrrolidone (PVP) in CSOF and transferred to a manipulation drop. The caudal fin of the egg donor was cut for genotyping analysis. PVP has been extensively used in human in vitro fertilization clinics and caused no detriment to zebrafish embryonic development (K.S., unpublished data). Preparation of donor cells. We prepared primary culture of adult cells from caudal fin. The fin was washed with LHC medium (Biosource, Inc.), disinfected in 0.04% bleach and rinsed using LHC. The fin was minced and transferred to a culture dish in D-NACs medium (Lin et al., 2005), containing DMEM supplemented with 2 mM N-acetyI-L- cysteine, 1 IIM ascorbate-Z-phosphate, 1% SeaGrow (East Coast Biolab, Inc.), 5% FBS and antibiotics. The explants were grown in 5% CO; with atmospheric air at 28 °C for over a month. Cells were freshly prepared before SCNT by trypsinization. A portion of cells were also kept for genotyping analysis. Embryo-derived cells were freshly isolated 86 from the tailbud of embryos at the 15—20 somite stage before SCNT. The tailbud was mechanically dissociated in D-NACs medium. The remaining part of the donor embryo was collected for genotyping analysis. All donor cells were aliquoted into original medium and kept at 4 °C until used for SCNT. Fresh donor cells were prepared in a drop of 2% PVP in serum-depleted medium every 2 h along the SCNT manipulation. Nuclear transfer. Nuclear transfer was performed using one pipette for holding the recipient egg and double injection needles for supporting and nuclear transfer. The egg holding pipette was cut straight and fire-polished to 200—300 um inner diameter. As the micropyle is very small and allows access of only a single sperm, 3 human intracytoplasmic sperm injection (ICSI) needle (Humangen) was used for nuclear transfer. For embryonic cells, needles with inner diameter of 5—6 pm were used. For cultured adult cells, needles with inner diameters of 8—9 pm were used. The supporting needle (inner diameter 20 um) was set up in parallel with the injection needle to help rotate the egg. Drops of 5% PVP in CSOF under mineral oil were used as manipulation medium. The x40 laser objective lens and controller (Hamilton Throne Bioscience, Inc.) were used to inactivate the egg genome. The egg was positioned with the micropyle facing the bottom of a manipulation dish, allowing the metaphase plate to be visualized best under UV light. The metaphase plate was burned twice using a laser beam (setting of 500 us with 100% power). Donor cells, placed in 2% PVP in serum-depleted D-NACs, were loaded into the lCSl needle, 3 process whereby the cell membrane was broken. The egg was repositioned with its micropyle now facing the injection needle, so that the donor nucleus and its remaining cytosol could then be transferred to the animal pole of 87 the eg via the micropyle. Approximately 5 eggs at a time were manipulated. The reconstructed embryos were washed in CSOF for 15 min and subsequently activated in egg water (60 jig/ml sea salt). The developmental potential of cloned embryos was monitored and recorded at blastula (3 h), germ ring (6 h), 90% epiboly (9—10 h) and 1- day to adult fish stages. To verify our manipulation technique, we produced zebrafish ICSI embryos by injecting sperm nuclei into 'off-target' laser-treated eggs (ablated location adjacent to the metaphase plate, sparing the egg's DNA). Approximately 5% fertile adult fish per total eggs manipulated were obtained using the ICSI technique (K.S., unpublished data). DNA fingerprinting. We selected SNP markers from the dbSNP database in Genbank based on chromosomal regions and a presence of restriction enzyme cutting site(s) both at the polymorphic nucleotide (diagnostic site) and, if possible, at the adjacent nucleotide (internal control site). We analyzed the genomic region of interest using UCSC genome browser (Kent, 2002) and designed primers using primer3 (Rozen and Skaletsky, 2000). SNP genotyping was analyzed by restriction fragment length polymorphism (RFLP) analysis after PCR. The SNP markers were tested and those found to be highly polymorphic among individuals were selected (Table 3). Genomic DNA of cloned embryos, donor cells and donor eggs was isolated using DNAeasy kit (Qiagen). Information regarding PCR, primers and restriction enzymes is available in Supplementary Table 1. PCR was done in 20 uL reaction mixtures containing 0.2 U Platinum Taq DNA polymerase (lnvitrogen), 0.5 pM each primer and 20—50 ng genomic DNA. The thermocycler program was 5 min at 94 °C, followed by 35—40 cycles of 94 °C 88 for 30 s, 55 °C for 30 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. The . PCR products were checked by 1% agarose gel electrophoresis and then digested with the restriction enzyme (NEB) at 37 °C overnight. RFLP was analyzed by 3% agarose gel electrophoresis using Ultrapure1000 (Invitrogen), except that SN P9 was analyzed by 6% PAGE (Table 3). Karyotyping and offspring production. Cultured cells derived from caudal fin of cloned fish were expanded and prepared for karyotyping as described above. Replication banding was chosen because it provides substantial resolution to identify different chromosomes of zebrafish (Amores and Postlethwait, 1999). Karyotyping of such cells was performed by Cell Line Genetics, LLC. Cloned fish at reproductive maturity were allowed to breed naturally with either homozygous golden or transgenic Tubingen long fin counterparts. The phenotype of the offspring was recorded and their reproductive soundness was evaluated. 89 Figure 18 Protocol for SCNT. Eggs were collected in CSOF and stained with Hoechst 33342. Laser-assisted XYClone module (Hamilton Throne Bioscience, Inc.) was used for targeted ablation of the metaphase plate of the recipient egg. The donor cell is gently broken and transferred through the micropyle. The reconstructed embryo is washed in CSOF, activated in egg water and raised at 28 °C. ._M_. Egg collection Laser targeted and DNA staining ablation of egg DNA Cell injection through micropyle I . O O o .H—...‘_‘ K. ~\,._ _\ f/ Wash in CSOF Donor cell preparation 1 Activate in egg water and raise embryo at 28 "‘C 90 Figure 19 Recipient eggs. Matured-arrest eggs were held in CSOF, and image was captured at 1 hour post-collection (a). Inset in (a) depicts DNA stained with HOECHST in a fixed egg which remained at metaphase ll of meiosis. (b) Nuclear staining of parthenogenetically activated eg at 20 min after egg-activation indicates female pronuclear formation and complete extrusion of second polar body — arrow (outside of the plane of focus). (c) In contrast to (b), nuclear staining of parthenogenetically activated egg at 20 min after laser-ablation shows complete inactivation of egg DNA and no extrusion of second polar body. Scale bar is 10 um, otherwise indicated. 91 Figure 20 Phenotype of cloned zebrafish and its offspring. (a) Brightfield image of a cloned embryo at 2 d of age showing golden pigmented pattern. Inset, an image of a wild-type fertilized embryo of same age. (b) Images of adult golden cloned fish showing lack of heavy pigments (bottom) and of wild-type pigmented female egg donor (top). (c) Fluorescence image of cloned 3-d golden embryo (bottom; no green fluorescence) and an in vitro—fertilized embryo of the H2AzGFP egg donor (top). (d) Brightfield image of the offspring of female cloned zebrafish and golden male that inherited the golden phenotype. Scale bars, 0.5 mm (a,c,d) and 0.5 cm (b). 92 8208 $0 8. .3: 00 0e0<<00<200... .050 50.. 0000.00. 0.02, 0:00 .0000 .34.... 0000 0030.030 90.... .0 .<~I. mmmo 3.09%: 0.00300: ......s. 030 0030...... 00.0: 005.0...00 0.0.5 000.00.000 520m 0 0 .0003... .303: .303qu 00000003 800050.00 .0« 0 0.2.3. 03 .3300 30000.0 £000.02 .3032... 8.0.05.3. «« 8.3.3.0.". 30300.0. me. m E. 03.05. .0.0 .0.0 .0.0 .303. 00 .303. m. .3000. 00 ...«030. 00 02 A ..< $000. <0. .0.0 6.00.0.0: 0.000.002 3.000.023 8000.003 8.0004300 8.000.300 03 m E. 00500.5: .0.0 .0.0 .300... H .0000... « .0000... 0 3000.0: 2 .3030. me 02 m ..< 50.00. ts .3000. « $000.0. 0 .0003. e .0003. A .0003. 0 $0000: 2 .0003... 3 000 m E 000.00. ts 3.003 0.5 .000. 0..... A$0.0 0 >00 H 200.3080 L3... .500 0.5.0.0 000.00.000 .00030000 3.00 «50.05000 .53 00 .0050: n 0 05.0.0 ... 003050 00 0500.0.“ 0005* __0._.0_,...Wu0 00 “Hymaz .“NQHHMM 00000 00000.00. 00.0000. 000350 .0 .0050: .000... .0 .00502 00200.0 0000.0 .0 000500.050 ”01.... 0.. ... 96 Figure 21 Abnormalities observed in cloned embryos. (a-b) Abnormal cloned embryos derived from embryonic cells at 1 day and (c) 3 days of age, (d-f) and cultured adult fibroblasts at 1 day of development. Scale bar is 0.5 mm. Most of the abnormal cloned embryos showed severe abnormalities of posterior development, and mild to moderate changes of anterior parts. \' a *‘ _. ‘~ . r: .' /’1'~ 97 Figure 22 Karyotyping and genotyping of cloned fish. (a) Karyotype analysis of cloned zebrafish by replication banding. Cloned zebrafish possessed normal diploid karyotype (2n=25). (b) Single nucleotide polymorphism (SNP) markers for genotyping analysis of cloned zebrafish by Restriction Fragment Length Polymorphism following polymerase chain reaction. A total of eleven informative markers are shown. Blue and red letters indicate genotypes. Arrows point to diagnostic bands of each genotype. IC is internal control for restriction enzyme activity. Of eleven SNP markers tested, we found a complete matched genetic identity between the donor cell and the cloned fish, no matching with the recipient egg donor was observed (Table 4). . I! ‘ I ‘ I \ f’.’ i l l l r? 1 2 3 4 s O ' g a ll Yl I: 77 %f 5 7 8 9 10 ‘1 i’ it i i )3 ‘c 11 12 13 14 15 i; 2' 5 J l, ‘15 } 16 17 18 19 20 3 3 ( < ! ! 7 4‘ '2 f a 21 22 23 24 25 (36 GA AA GA AA CG CG CC CC TT CC TC > unal- . 00M. an > H . '° J. - tn. . .. '...~’.- to $095 snps . ’ SNP3 AA cc CA 0c 00 cc - > p > H: :2 I 0‘ u “we . '53:” ’ sums 98 CHAPTER 5 INFLUENCE OF DONOR NUCLEUS SOURCE IN THE OUTCOME OF ZEBRAFISH CLONING PROCEDURES Kannika Siripattarapravat, Boonya Pinmee, EunAh Chang, Juan David Munoz, Koichi Kawakami, and Jose 8 Cibelli Abstract The donor cells from five different tissues of transgenic zebrafish were compared for their capacity to be reprogrammed following somatic cell nuclear transfer. Donor cells of the HGZlC line, cells of fin and notochord origin, gave the best rate of cloned fish production. While cells from other lineages were tested and, indeed produced cloned fish, the efficiency of cloning was significantly lower than the ones selected from the HGZlC fish line. Introduction It is known that as a cell differentiates, its developmental potential gets more restrictive. Following somatic cell nuclear transfer (SCNT), cells can be reprogrammed to an embryonic state at different efficiencies depending on the type of cell or tissue origin. We have learnt from mouse experiments that the easiest cells to reprogram are blastomeres from a morula, however the efficiency progressively declines when cells from the inner cell mass and tissue specific cells are used (Gurdon and Melton, 2008; Hochedlinger and Jaenisch, 2006; Thuan et al., 2010; Wakamatsu, 2008). Side by side comparisons of different cell types were made by the same laboratory and found that ESCs are more amenable to cloning than somatic fibroblasts (Rideout et al., 2000) and 99 even among different somatic cell populations, the variations of cloning efficiency are significant (Oback, 2009; Oback and Wells, 2007). Adult frogs were obtained when donor nuclei was isolated from blastomeres (Gurdon et al., 1958) and embryonic- intestinal cells (Gurdon and Uehlinger, 1966). But only tadpoles were produced using adult cells as donor nuclei (Gurdon et al., 1975; Laskey and Gurdon, 1970). These evidences point toward the existence of a cell to cell variability that can be attributed to the epigenetic state that defines the phenotype of a given cell. Effective nuclear reprogramming requires shutting down somatic—cell specific gene expression and turning on embryonic-specific genes in a carefully choreographed manner. In SCNT, errors of the nuclear reprogramming were observed in almost all species cloned (Cibelli et al., 2002). In mice, when muscle cells were used as donor nuclei, the GLUT4 glucose transporter gene continued to be active in the early cloned mouse embryos (Gao et al., 2003). Furthermore the pluripotentcy-related gene, Oct 4, is expressed incorrectly in the majority of the cloned embryos produced using nuclei from cumulus cells (Boiani et al., 2002). In frogs, tissue-specific gene expression of a donor cell was found to persist in cloned embryos, a phenomenon known as epigenetic memory (Ng and Gurdon, 2005; Ng and Gurdon, 2008). Despite these abnormal patterns of gene expression, a small population of cloned animals can develop into seemingly healthy adults. It has been suggested that one of the most important steps towards successful SCNT is the selection of a donor population of nuclei that are intrinsically more reprogrammable by the recipient oocyte (Santos and Dean, 2004). There are evidences 100 indicating that the cell donor is responsible for variations in the efficiency of SCNT (Kato et al., 2000; Wakayama and Yanagimachi, 1999), yet it is inconclusive (Oback, 2009). Work done in our laboratory has shown that in zebrafish, the efficiency to produce cloned hatched-fry can be 643% when donor cells are freshly isolated from embryonic tail-bud (Siripattarapravat et al., 2009b). Based on the evidence, we hypothesized that nuclear reprogramming efficiency in zebrafish varies from one tissue-specialized cell to another. In other words, cells from different lineages could have different developmental potential when used as donor cells for SCNT. To test this hypothesis, we proposed to evaluate the efficiency of cloning zebrafish (measured by reconstructed embryos that develop to normal hatched-fry stage) when donor cells derived from three different sources, ectoderm, mesoderm, and endoderm, were used. We used transgenic zebrafish expressing green fluorescence protein (GFP) under a very ’tight’ endogenous tissue specific promoter (Figure 23) (Nagayoshi et al., 2008). All transgenic lines were generated with the to! 2 vector system developed by the Kawakami lab (Nagayoshi et al., 2008). These fish express GFP in tissue specific manner, approximately at 24 hpf, allowing for a rapid-live cell type identification. We report here the cloning efficiency of 5 different cell types in Zebrafish. We used 5 different transgenic lines: 1) HGn62A-skin (ectoderm), 2) HGn28A—skin (ectoderm), 3) HGnBE—heart (mesoderm), 4) HGZlc—fin/ notochord (mesoderm), and S) HGn30A-hatch gland (endoderm) (Nagayoshi et al., 2008). The result showed that GFP+ 101 cells from lines HGn21C are much more amenable to nuclear reprogramming than the others. 33.5255. SCNT was performed as previously described (Siripattarapravat et al., 2009b), with the exception that the donor cells were from embryos 24 hours post-fertilization (hpf) and were selected for GFP+ prior to nuclear transfer (Figure 23). We observed devel0pmental capacity of cloned embryos derived from donor cells of different sources (Table 6 and Figure 24). All cell types used yielded cloned embryos albeit at different rates of developmental capacity and degree of normality. We were able to clone adult zebrafish from donor cells of HGn28A and HGn8E. Following back-crossed with the wild type strain, the offspring of these clones were normal and carried lineage specific GFP+ as their cloned parental lines (Figures 23F and 236). All cell types from the different transgenic fish line had different sizes, morphology, and appearances. GFP+ cells of HGn28A and HGZlC lines were the smallest, and just fit the needle with a diameter of 7-8 pm. The cytoplasmic membrane of both cells was easily broken in the injection needle. GFP+ cells of the HGn62A line, whose cytoplasmic membrane is not easily broken, were the largest and only fit in the injection needle with diameter of 9-10 pm. All of the GFP+ cells from HGn28A, HG21C, and HGn62A lines were abundant in the embryos and easily identified in the manipulation drop, likely shortening the amount of time that the cells were exposed to the UV light when compared to HGn30A and HGn8E lines. GFP+ cells of the HGn30A line were large and contained multiple cytoplasmic vesicles. Though cells were big in size, they were 102 very fragile as they broke at suction and could be squeezed easily through an injection needle with diameter of 8 pm. GFP+ cells of the HGn8E line were of medium size with a cell membrane which was very elastic and had to be forced through the needle a few times prior to disruption of the cytoplasmic membrane. It remains to be determined if all these physical characteristics of the cells have any impact on the overall efficiency of SCNT, however for the purpose of this study, we assumed that the most important determining factor is the epigenetic status of the cell nucleus. The developmental rates of cloned embryos were recorded at the blastula stage, germ ring (entering gastrulation), 90% epiboly (complete gastrulation), day 1 (complete segmentation), day 4 - hatched fry, eating fry, and adult (Kimmel et al., 1995). Cloned embryos were classified according to their morphology and recorded as either normal embryos or total embryos (including abnormal counts). Results are shown in table 6 and figure 24. At the blastula stage, cell division is the major event with cells undergoing approximately 10-11 cell divisions before entering mid-blastula transition, when the cell cycle is no longer homogeneous and lengthens; at this point zygotic gene transcription starts (Kimmel et al., 1995). Except 18% in HGn8E, approximately 40% of cloned embryos completed development to normal blastula. The abnormality of cloned embryos recorded at this stage was partial blastula embryos. At gastrulation stage, the cells start differentiation, migration, and form the germ ring (GR). Only 5% of cloned embryos from HGn62A, HGn30A and HGn8E developed to normal GR, while observing 12% of HGn28A and 20% of HGZlC. The abnormal cloned embryos at GR showed unequal migration of the cells toward vegetal pole of the egg. Upon finishing 103 gastrulation, at 90% epiboly (EB), cells have completely migrated from the animal pole to vegetal pole, and the three germ layers are formed. Except 16% of HGZIC, only 3-5% of cloned embryos made to normal EB. Most of abnormal cloned embryos at EB stage showed lower cell density than the normal embryos at this stage. The segmentation period is followed, and as cells progress in differentiation they form somites, and start organogenesis. By day 3, the embryos finish organogenesis, hatch from the chorion, and develop swim bladder. At day 1 to 4, the number of cloned embryos dropped dramatically to 0.4% in HGn62A and HGn30A. For HGn28A and HGZlC, the number of cloned embryos remained at 2-3% in day 1, and dropped by half at day 4. The cloned embryos of HGn8E remained at 1.6% until day 4. As previously reported (Siripattarapravat et al., 2009b), we observed various degrees of abnormalities in cloned embryos at 1-4 days. All of the normal cloned embryos at day 4 started to eat. Only two fry from GFP+ donor cells of HGn28A and HGn8E lines were made to adult fish A two-way analysis of variance was used to analyze the data. The model was set for binomial distribution of counting dataset under PROC GRIMMIX (SAS system). Since the variances are discrete in 2 developmental stages from others, i.e., the number of eating fry and adult, they were excluded from the statistical analysis. Statistical analysis has shown no significant interaction found between sources of GFP+ donor cells and numbers of cloned embryos at all developmental stages recorded. When contemplating donor cell individuals and accounting for numbers of total embryos counted from all developmental stages (from blastula to 4 days), cloning efficiency from HGZ 1C donor cells was significantly higher than any other donor cells (p 104 < 0.05). When comparing numbers of normal embryos, no difference was found among donor cells of all lineages. 00.110192 We have found that the developmental capacity between cell lines was different when analyzed at blastula to hatched fry stage. All cell lines were capable of generating cloned fish with the HGZIC line being the most efficient. A significantly large number of cloned fish failed to develop normally most likely due to failures of reprogramming. We speculate that no altered phenotypes were due to the transgene present in the fish lines used since four out of the five lines harbor the inserted gene in the intronic region of the genome, and while the HGZlC line has its transgene inserted into the tcf 7 gene (transcription factor 7) sequences, only the homozygous mutants show abnormal development of fins. A potential confounding factor that could have negatively impacted the rate of normal fish generated, could be the use of UV light to locate the DNA in the egg nucleus and to select transgenic cells. However, three pieces of evidence argue against such speculation. First, we ran control experiments in which eggs were exposed to UV light and their cytoplasm laser irradiated followed by in vitro fertilization, and healthy fish were generated after this manipulation. Second, our method explicitly minimized UV exposure to less than five seconds; and third, fresh donor cells were freshly loaded every 30 minutes. Regardless, if there was a potential for increasing the rate of abnormalities due to UV irradiation, all experiments would be equally affected since all five transgenic fish lines were subjected to the same treatment. Taken together we can 105 conclude that UV exposure should not be a confounding factor in the overall efficiency comparison between treatments. it has been demonstrated in several mammalian species that SCNT is possible with donor cells are at either 61/60 or GZ-M, but not S phase (Campbell et al., 1996; Cibelli et al., 1998; Egli et al., 2007; Wakayama et al., 1999). We did not test the cells for their stage in the cell cycle prior to SCNT, and it is possible that the cells used as donors were at different stages in the cell cycle. However, in an attempt to standardize our protocol, we purposely selected cells that were the smallest in the pool, likely selecting only cells in the GO to 61 stage. Nonetheless, more work is needed to determine whether cells at different stage of cell cycle will have different cloning efficiency in zebrafish. GFP+ cells in the HGn30A and HGnBE lines are distinctly Specialized cells. Cells from the HGn30A line contain multiple cytoplasmic vesicles, possibly storing proteolytic enzymes produced from the cells of the hatch gland. The injection technique was done by delivery of all cytoplasmic components together with the nucleus at the time of nuclear transfer. It is possible that the components in those vesicles have negative impact onto the reconstructed embryos and the capacity for nuclear reprogramming. GFP+ cells of HGn8E line are heart muscle cells, some of them are multinucleated cells. It is possible that more than one nucleus was transferred and that may have caused ploidy abnormalities in the cloned embryos. In addition, muscle cells are known to continue to express muscle specific genes, possible making them more resilient to epigenetic modifications (Gao et al., 2003). 106 In mouse cloning, the abnormality observed in cloned mice was not only due to epigenetic reprogramming, but also the karyotypic abnormalities from manipulation of donor nucleus (Wakayama and Perry, 2002). We have not analyzed for karyotypes in the abnormal clones embryos therefore we cannot rule out all possible causes of abnormalities in the clones. in summary, we found that GFP+ cells isolated from the HGZlC zebrafish line yield the highest capacity for nuclear reprogramming following SCNT. A thorough analysis of the epigenetic signatures of these cells may help us elucidate specific factors that are responsible for their enhanced reprogramming capacity. In a more practical application, we expect to use these cells for gene targeting experiments in vitro followed by SCNT. In turn, this work could facilitate the generation of generation of knock-in/ -out zebrafish founder animals providing the zebrafish research community with an unparalleled tool for studies of gene loss/gain of function. Methods Zebrafish strain. The outcrossed between Tubingen and AB line, named TAB, was used as female egg donors. The transgenic zebrafish (Nagayoshi et al., 2008) with Tubingen long-fin background, expressing tissue specific green fluorescence protein (GFP), HGn30A-hatch gland, HGn28A and HGn62A-skin, HGZlc-fin/notochord, and HGn8E- heart were used to isolate donor cells. Preparation of recipient eggs and donor cells. The recipient eggs were obtained by stripping technique (Westerfield, 1993). The eggs were immediately placed in Chinook salmon ovarian fluid (CSOF)(Siripattarapravat et al., 2009a). The eggs were stained with 107 50mg/ml Hoechst33342 for 20 minutes as described previously (Siripattarapravat et al., 2009b), and kept in CSOF until used for nuclear transfer. The donor cells were freshly prepared from embryos at 24 hour post-fertilization. The embryos were sorted for GFP positive under fluorescence microscope. For HGn30A, HGZlc and HGn8E, the embryos were dissected and selected for the GFP positive tissues. For HGn28A and HGn62A, the whole embryos were extracted from the yolk prior to use. Subsequently, embryos were briefly minced in LHC basal media, and trypsinized (with 0.025% trypsin) at room temperature for 10—15 minutes. The activity of trypsin was then inhibited by using 5% fetal bovine serum in LHC. The cells were washed twice with LHC, and kept in DNACs medium (Siripattarapravat et al., 2009b) until used for nuclear transfer. In case of HGn30A and HGn8E, more than 20 embryos were utilized in each manipulation as there were limited numbers of GFP expressing cells in each embryo. For other strains, only 5- 10 embryos were used. The cell suspension was added to a new drop in manipulation dish every 30 minutes to minimize repeated UV exposure of donor cells. Somatic cell nuclear transfer. The nuclear transfer was performed as described previously (Siripattarapravat et al., 2009b), with minimal modifications. For enucleation, the DNA-stained egg’s metaphase plate was ablated within the chorion using laser firing. The injection needle 8 pm in diameter was used for all cell types, except HGn62A in which the injection needle with 9 pm in diameter was used. Prior to injection, the cells were selected for GFP expression and individually picked for nuclear transfer. The individual donor nucleus was then transferred to the eg through the micropyle (the sperm entry site). Reconstructed embryos were activated in ’embryo medium’ 108 (Westerfield, 1993) and allowed to develop. The developmental potential of cloned embryos were monitored and recorded every 3 hours after egg activation to 1 day, and continued every day until adulthood. Statistical analysis. Developmental potential of cloned embryos was analyzed statistically by using two-way ANOVA and testing for the effects of two factors: the types of donor cells and the number of live embryos at 5 developmental stages. Since the response variable was the number of living embryos from the initial total reconstructed embryos, we considered modeling the count nature of the data assuming a binomial distribution under PROC GLIMMIX (SAS Institute, 2008). Repeated measures analysis was considered for developmental stages which required the modeling of a covariance structure across time points. Autoregressive model for covariance structure was preferred based on Akaike's information criterion values. Least squares means were estimated after back transforming from the binomial distribution using a logistic link function. Means for percent of embryos obtained from each type of cells were compared for significant difference within each developmental stage using Fisher’s protected LSD and alpha<0.05. 109 Figure 23 GFP+ donor embryos and offspring of cloned zebrafish. Tissue specific GFP+ embryos at 24 hours post-fertilization of HGn30A (A), HGn62A (B), HGZlC (C), HGn28A (D), and HGn8E (E) that are sources of GFP+ donor cells (Nagayoshi et al., 2008). Back- crossed with wild type strain, offspring of cloned zebrafish from HGn28A GFP+ donor cells (F) and HGn8E GFP+ donor cells (G) of the same age. Scale bar is 250 um. 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