Avian Germplasm Preservation: Embryonic Stem Cells or Primordial Germ Cells? 1

Transcription

1 Avian Germplasm Preservation: Embryonic Stem Cells or Primordial Germ Cells? 1 J. N. Petitte 2 Department of Poultry Science, College of Agriculture and Life Sciences North Carolina State University, Raleigh ABSTRACT Presently, avian genetic resources are best cells, ESC, or PGC might offer a means to preserve the maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with destroyed because of the decline in the number of Poultry varying degrees of efficiency. Similarly, the effectiveness Science Departments and pressures to cut costs at land of generating germ line chimeras using blastodermal cells, grant institutions. Cryopreservation of semen is often ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the not allow for full reconstitution of the genetics of the low efficiency rate of reconstitution and the high cost original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primorproaches prohibitive. Significant challenges remain to be associated with current technologies makes these apdial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted. Key words: genetic conservation, primordial germ cell, chicken, embryonic stem cell, germ cell 2006 Poultry Science 85: INTRODUCTION To those who work with poultry, whether in industry or academia, the value of avian genetic resources has been immeasurable. Highly selected, specialized populations have been developed that have contributed to advances in biology, medicine, and agriculture for over half a century. Today, a large global industry is dependent upon genetic diversity for the efficient and inexpensive production of meat and eggs. However, during the last 3 decades, public institutions have seen a dramatic drop in the number of research lines available. Ironically, the decline in poultry genetic resources comes at a time when the release of the draft sequence of the chicken genome promises a new wealth of knowledge and benefits (Fulton and Delany, 2003). With a decline in living stocks, it is now more important than ever to use current methods and to develop new methods to preserve avian germplasm. Cryopreservation 2006 Poultry Science Association, Inc. Received October 17, Accepted October 17, Paper for the Poultry Science Association Ancillary Scientists Symposium, July 31, 2005, Auburn, Alabama, Conservation of Avian Genetic Resources: Current Opportunities and Challenges, organized and chaired by Muquarrab Qureshi. 2 Corresponding author: j_petitte@ncsu.edu of semen has been used and will continue to be a part of a multifaceted strategy to preserve genetic variation. Semen cryopreservation works well for single-gene traits. However, it cannot be used to store and reconstitute highly inbred or specially selected lines of poultry, as only the male genome is represented. Therefore, other methods of germplasm preservation are needed. Over the last 15 yr, there has been considerable interest in the manipulation of the early embryo using avian embryonic stem cells (ESC) and primordial germ cells (PGC) for the production of transgenic poultry. This work has lead to a number of technologies that could be used for the preservation of avian germplasm. The purpose of this review is to examine some of these technologies and to evaluate their potential for the cryopreservation and reconstitution of rare genetics stocks. THE GERM CELL CYCLE If the full genetic complement of an endangered stock is to be preserved, it is necessary to preserve the germ line. The germ cell cycle begins with fertilization. Like all vertebrates, the avian germ line is established in an extragonadal location early in embryonic development. Subsequently, the germ cells migrate to the germinal ridge and proliferate during the first week or so of embryo development. Male germ cells do not begin substantial levels of proliferation until sexual maturity when the pro- 237

2 238 PETITTE cess of spermatogenesis begins, leading to the development of mature sperm. In females, the oocytes undergo meiotic arrest shortly before or after hatch and remain quiescent until sexual maturity when oocyte growth and maturation takes place. Subsequently, the germ cell cycle is repeated with fertilization. For the purposes of cryopreservation, it is necessary to target germ cells between fertilization and the beginning of sexual differentiation. One of the main characteristics of reproduction in birds is the oviposition of a hard-shelled egg containing a large yolkly ovum surrounded by layers of albumen. After ovulation, the ovum is captured by the infundibulum of the oviduct, where fertilization takes place. Fertilization in birds is polyspermic, and many sperm nuclei can be found after penetration of the vitlletine membrane; however, only one sperm will fertilize the egg (Perry, 1987). Subsequently, the fertilized egg enters the magnum, where a firm albumen capsule encases the ovum. Later, as it transverses the isthmus, the outer and inner shell membranes are laid down in preparation for deposition of the eggshell. The first cleavage divisions occur upon entry of the ovum into the shell gland. The egg spends the longest time in the shell gland, taking 20 to 22 h to complete formation of the eggshell. A rapid cell division occurs during eggshell formation, and the embryo acquires its polarity, e.g. anterior/posterior, yet it is visually, radially symmetric. When the egg is laid, the diskshaped embryo or blastoderm contains about 50,000 to 60,000 cells lying on the surface of the yolk. At this time, the blastoderm can be divided into a peripheral ring of cells attached to the yolk (called the area opaca) and a central, more translucent region (the area pellucida). The area pellucida is suspended above a non-yolky fluid deposited by the embryo. This arrangement is advantageous and allows for the easy manipulation of the embryo. In general, the area opaca will contribute only to extraembryonic structures. Upon incubation, the area pellucida differentiates into 2 layers, an upper epiblast and a lower hypoblast. Only the epiblast will give rise to the embryo proper while the hypoblast contributes to some extraembryonic tissues. This period of development from fertilization through hypoblast formation has been classified into a series of 14 stages by Eyal-Giladi and Kochav (1976) for the domestic hen and are indicated using Roman numerals. Subsequent stages are classified using the staging system of Hamburger and Hamilton (1951) using Arabic numerals. The next period of embryo development that is significant for germplasm preservation of poultry is the establishment of the germ line. Definitive PGC were identified almost a century ago, when Swift (1914) described PGC in an extraembryonic region called the germinal crescent. The germinal crescent lies in an anterior region formed during gastrulation, as the hypoblast is displaced by the endoderm. Swift s identification of the germinal crescent was based on the morphological characteristics of PGC. From the germinal crescent, the germ cells must find their way to the developing gonadal ridge. As the blood islands form and the intra- and extraembryonic vasculature develops, the PGC are carried to the vicinity of the germinal ridge through the embryonic circulation (Swift, 1914; Meyer, 1964; Fujimoto et al., 1976a,b). Subsequently, the blood-borne PGC actively leave the vessels and migrate to the germinal epithelium through the dorsal mesentary. Once the germ cells arrive in the primitive gonad, sexual differentiation begins. CONSIDERATIONS FOR CRYOPRESERVATION For cryopreservation of the germ line to be successful, a ready source of germ cells is needed, and efficient means of reconstituting the line are required. Figure 1 sets out the required elements in a technological scheme to make cryopreservation feasible. First, cells must be obtained from embryos. These can be freshly isolated blastodermal cells from Stage X unincubated embryos or PGC obtained from Stages 14 to 16 during their migration in the embryonic circulation or at about Stages 26 to 28 when they settle in the germinal ridge. Because most threatened genetic stocks do not exist in large numbers, some means of amplifying the number of cells through culture would be ideal. Once the cells are obtained, germ line chimeras could be produced through the transfer of cells at the appropriate stage of development. The manipulated eggs would need to hatch, and then the resulting chimeras could be mated to each other to provide identifiable offspring that would reconstitute the original line. Three sources of cells have been proposed for cryopreservation: blastodermal cells, ESC, and PGC. BLASTODERMAL CELLS Germ line chimeras can be produced using freshly isolated cells from the Stage X embryo. Petitte et al. (1990) injected dispersed Stage X blastodermal cells into the subgerminal cavity of unincubated embryos and obtained a rooster that was both a somatic and germ cell chimera. Although the efficiency of germ line transmission was about 0.3%, this stimulated a considerable amount of work related to the development of chimeras using fresh blastodermal cells. Germ line chimerism using blastodermal cells can depend on the lines used as a donor and recipient (Petitte et al., 1993; Thoraval et al., 1994). Cells should be taken from the central disk of the area pellucida, and the donor and recipients should be developmentally matched (Petitte et al., 1993). Mixed-sex chimerism can be problematic; therefore, only same-sex chimeras should be produced (Shaw et al., 1992; Kagami et al., 1995, 1997). Lastly, and perhaps most importantly, the efficient of production of germ line chimeras using blastodermal cells requires compromising donor embryos with a sublethal dose of 500 to 600 rads of gamma radiation (Carsience et al., 1993) or through the removal of about 700 cells from the central part of the area pellucida of recipient embryos and replacing the same location with donor cells (Kagami et al., 1997). Even with all of these considerations, the high

3 ANCILLARY SCIENTISTS SYMPOSIUM 239 Figure 1. Diagram of the steps required for the cryopreservation of poultry genetic stocks and their reconstitution using chimeras. Cells from the Stage X embryo or primordial germ cells (PGC) can be removed and cryopreserved. Alternatively, the blasotermal cells can be cultured as embryonic stem cells (ESC) or as PGC prior to cryopreservation. To reconstitute the line, thawed cells are injected into recipient embryos. Donor cells should be injected into recipient embryos of the same stage of development. The resulting male and female chimeras can be mated to produce donor-derived offspring with the complete donor genotype. efficiency of germ line transmission is not guaranteed for every individual bird. Recently, Bednarczyk et al. (2002) reported the reconstitution of a breed of chicken apparently native to Poland, viz. Green-legged Partridgelike (GP). The recipient was a White Leghorn (WL) line. Twenty-three putative WL/ GP chimera hatched, and 20 survived to sexual maturity. All putative chimeras were test-mated for germ line transmission using crosses with GP birds. Of 10 males and 10 females, 2 males were chimeras and transmitted at 17.6 and 50%; 4 of the females transmitted at 5, 20, 23, and 23%. When the male transmitting at 17.6% was mated with all 4 females, GP offspring were obtained only from one female. In total, 696 eggs were set, 477 chicks hatched to yield only 10 GP chicks. A DNA polymorphism analysis of the chicks indicated a significant amount of variation among the resulting GP birds. Although this study suggests that it is possible to reconstitute a breed of chicken using blastodermal cell chimeras, significant barriers for its use for routine conservation of endangered poultry stocks remain. Naito et al. (1992) demonstrated that frozen blastodermal cells from quail embryos could be used to make quail/chick somatic chimeras. However, when chicken blastodermal cells were frozen in dimethylsulfoxide and thawed to produce germ line chimeras, the thawed cells needed to be purified using density centrifugation (Kino et al., 1997). This processing of the blastodermal cells resulted in 5 germ line chimeras of the 53 chimeras produced. The rate of germ line transmission was about 6%, much lower than the 29.5% observed using fresh blastodermal cells. Before blastodermal cells can be used to preserve genetic lines, procedures are needed for making high-grade consistent germ line chimeras and for efficient cryopreservation of blastodermal cells. ESC Embryonic stem cells are pluripotent cells that can, by definition, give rise to all tissue types, including the germ line. Avian ESC have been cultured from the Stage X embryo with varying degrees of success (Pain et al., 1996; Petitte et al., 2004; Zhu et al., 2005). Avian ESC can be cultured, frozen, and used to make somatic and germ line chimeras. Even high-grade somatic chimeras can be

4 240 PETITTE made using embryonic stem cells (Zhu et al., 2005). However, the main impediment to their use for banking avian germplasm is the low efficiency of germ line transmission. Presently, the ability of the cells to give rise to the germ line is lost with after culture for more than a few weeks (Pain et al., 1996; Petitte et al., 2004). This feature alone makes avian ESC unsuitable at this time. PGC Three sources of PGC have been used to produce germ line chimeras, the germinal crescent, germ cells circulating in the embryonic blood, and germ cells harvested upon arrival in the primitive gonad. Reynaud (1969) was the first to show that PGC from the germinal crescent could be injected into the blood vessels of recipient embryos and settle in the host gonad. Wentworth et al. (1989) reported that quail germinal crescent PGC could be used to generate germ line chimeras. However, the germinal crescent is the least useful source of PGC for cryopreservation given the low numbers of germ cells during this period of germ cell development (Stages 4 to 8). Simkiss et al. (1989) demonstrated that circulating chick PGC could be used as donor cells and migrate to the gonad. Subsequently, Tajima et al. (1993) successfully hatched germ line chimeras after the transfer of blood PGC to recipients of the same stage of development. Gonadal PGC can also be used to generate germ line chimeras. It is now accepted that PGC obtained from the gonad of 5- to 7-d-old embryos can actively migrate to the gonadal ridge in embryos during the period of circulation in the blood, i.e., Stages 14 to 17 (Tajima et al., 1998). Wentworth and Wentworth (2000) reported a similar phenomenon in quail. As observed with the production of blastodermal cell chimeras, some type of compromising of the recipient embryo is needed. When endogenous PGC are removed from the embryonic circulation prior to injecting donor blood PGC, the level of germ line transmission increased (Naito et al., 1994b). The number of endogenous PGC can also be reduced using an injection of busulphan (Aige- Gil and Simkiss, 1991; Song et al., 2005) and can also lead to better germ line transmission rates (Vick et al., 1993; Song et al., 2005). Along with reducing the number of endogenous PGC, attempts have been made to concentrate the number of donor PGC obtained from embryonic blood using Ficoll or Nycodenz density gradient centrifugation (Yasuda et al., 1992; Zhao and Kuwana, 2003) and immunomagnetic cell separation (Ono and Machida, 1999; Wei et al., 2001). Recently, pure populations of PGC were obtained from embryonic blood and embryonic gonads using fluorescence-activated cell sorting purification (Mozdziak et al., 2005). Given that most lines targeted for cryopreservation will have limited numbers of birds, it would be advantageous to be able to culture PGC prior to cryopreservation. A few studies have attempted the culture of PGC from blood or gonads, but these have been limited to short-term culture. Chang et al. (1995a,b) cultured PGC from blood for about 5 d and produced germ line chimeras (Chang et al., 1997). Karagenc and Petitte (2000) were able to stimulate PGC proliferation from blastodermal cells. Han et al. (2002) reported the culture of gonadal PGC for up to 2 mo that remained germ line competent. Interestingly, Park et al. (2003) reported that the culture of gonadal PGC for 10 d enhanced their ability to generate germ line chimeras. The long-term culture of PGC that were germ line competent would provide useful tools for cryopreservation of genetic stocks. In a few cases, cryopreservation of PGC have been reported. Naito et al. (1994a) collected circulating PGC from Stages 13 to 15, concentrated the PGC using a Ficoll gradient, and froze them in dimethylsulfoxide. Germ line chimeras were produced by injection of 100 thawed PGC. Viable offspring were obtained through inter se matings. Tajima et al. (1998) demonstrated that cryopreserved gonadal PGC retained the ability to generate germ line chimeras. However, in all cases, the level of germ line chimerism was low, making the process inefficient for routine use. OTHER BARRIERS TO EFFICIENCY In addition to the choice of cells for cryopreservation and the efficiency of chimerism, 3 other factors need to be considered when examining the use of chimeric intermediates as a means of preserving genetic resources. First, the physical manipulation of the avian egg presents some technical difficulties of its own. If blastodermal cells are going to be used to generate germ line chimeras, methods for manipulating the avian egg can require specialized procedures to ensure that the injected embryos will hatch. The culture of unincubated embryos in surrogate eggshells successfully produced live birds that reached sexual maturity (Ono and Wakasugi, 1984; Rowlett and Simkiss, 1987). In 1988, Perry developed a complete culture system bringing the chick embryos from the single-cell stage to hatching. However, Perry (1988) required 3 different sequential culture systems and yielded 7% hatchability. Subsequently, culture procedures have been developed to improve hatchability to 34% (Naito et al., 1990; Naito and Perry, 1989), and more recently, surrogate eggshell culture of unincubated embryos resulted in 75% hatchability (Borwornpinyo et al., 2005). Alternatives to surrogate eggshell culture involve simple windowing (Bednarczyk et al., 2000; Speksnijder and Ivarie, 2000). Recently, an improved sealing technique to avoid trapped artificial air bubbles during sealing resulted in 45% hatchability (Andacht et al., 2004). Procedures appropriate for eggs injected at later stages of development when injecting PGC into recipient embryos involve simple windowing procedures. However, the hatchability of simple windows hovers around 50%. Hence, there is room for significant improvement. Second, selection of the recipient line to make chimeras could become extremely important in reconstituting a line from banked germ cells. Some recipient lines could be

5 ANCILLARY SCIENTISTS SYMPOSIUM 241 more suitable than others. Ideally, a universal recipient line would be one where the endogenous PGC were low and the genetics such that the donor offspring could easily be distinguished from the recipient. Not all endangered lines are pigmented, and the genetics of white-feathered lines could be unknown and problematic. Hence, identification of the donor germ line may require specialized screening procedures. Many factors impinge upon the final consideration, the cost of reconstituting the line. Implicit in the cryopreservation preservation of rare lines of poultry is the idea that a measure to allow reconstitution of the lines will be easy and inexpensive when needed by researchers or the industry. As the number of Poultry Science Departments decline, few institutions will have the facilities to regenerate interesting lines for research. However, unless reconstitution procedures are easy and inexpensive relative to the value of the line, few lines will ever be reconstituted and used except in crisis situations. CONCLUSIONS Much refinement in all aspects of the forgoing procedures is needed to fully utilize the potential to preserve avian genetic diversity using blastodermal cells, ESC, or PGC. To date, the best approach lies with either blastodermal cells or PGC. However, if PGC could be cultured to amplify the number of germ cells for cryopreservation, this would represent a major advance toward preserving the remaining poultry stocks that are continually under pressure to become extinct. REFERENCES Aige-Gil, V., and K. Simkiss Sterilisation of avian embryos with busulphan. Res. Vet. Sci. 50: Andacht, T., W. Hu, and R. Ivarie Rapid and improved method for windowing eggs accessing the stage x chicken embryo. Mol. Reprod. Dev. 69: Bednarczyk, M., P. Lakota, and M. Siwek Improvement of hatchability of chicken eggs injected by blastoderm cells. Poult. Sci. 79: Bednarczyk, M., P. Lakota, R. Slomski, A. Plawski, D. Lipinski, B. Siemieniako, M. Lisowski, P. Czekalski, B. Grajewski, and P. Dluzniewska Reconstitution of a chicken breed by inter se mating of germline chimeric birds. Poult. Sci. 81: Borwornpinyo, S., J. Brake, P. E. Mozdziak, and J. N. Petitte Culture of chicken embryos in surrogate eggshells. Poult. Sci. 84: Carsience, R. S., M. E. Clark, A. M. Verrinder Gibbins, and R. J. Etches Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117: Chang, I. K., D. K. Jeong, Y. H. Hong, T. S. Park, Y. K. Moon, T. Ohno, and J. Y. Han Production of germline chimeric chickens by transfer of cultured primordial germ cells. Cell Biol. Int. 21: Chang, I. K., A. Tajima, T. Chikamune, and T. Ohno. 1995a. Proliferation of chick primordial germ cells cultured on stroma cells from the germinal ridge. Cell Biol. Int. 19: Chang, I. K., A. Yoshiki, M. Kusakabe, A. Tajima, T. Chikamune, M. Naito, and T. Ohno Germ line chimera produced by transfer of cultured chick primordial germ cells. Cell Biol. Int. 19: Eyal-Giladi, H., and S. Kochav From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev. Biol. 49: Fujimoto, T., T. Ninomyiya, and A. Ukeshima. 1976a. Observations of the primoridal germ cells in blood samples from the chick embryo. Dev. Biol. 49: Fujimoto, T., A. Ukeshima, and R. Kiyofuji. 1976b. The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat. Rec. 185: Fulton, J. E., and M. E. Delany Genetics. Poultry genetic resources Operation rescue needed. Science 300: Han, J. Y., T. S. Park, Y. H. Hong, D. K. Jeong, J. N. Kim, K. D. Kim, and J. M. Lim Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 58: Hamburger, V., and H. L. Hamilton A series of normal stages in the development of the chick embryo. J. Morphol. 88: Kagami, H., M. E. Clark, A. M. Verrinder Gibbins, and R. J. Etches Sexual differentiation of chimeric chickens containing zz and zw cells in the germline. Mol. Reprod. Dev. 42: Kagami, H., T. Tagami, Y. Matsubara, T. Harumi, H. Hanada, K. Maruyama, M. Sakurai, T. Kuwana, and M. Naito The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken. Mol. Reprod. Dev. 48: Karagenc, L., and J. N. Petitte Soluble factors and the emergence of chick primordial germ cells in vitro. Poult. Sci. 79: Kino, K., B. Pain, S. P. Leibo, M. Cochran, M. E. Clark, and R. J. Etches Production of chicken chimeras from injection of frozen-thawed blastodermal cells. Poult. Sci. 76: Meyer, D. B The migration of primordial germ cells in the chick embryo. Dev. Biol. 10: Mozdziak, P. E., J. Angerman-Stewart, B. Rushton, S. L. Pardue, and J. N. Petitte Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult. Sci. 84: Naito, M., K. Nirasawa, and T. Oishi Development in culture of the chick embryo from fertilized ovum to hatching. J. Exp. Zool. 254: Naito, M., K. Nirasawa, and T. Oishi Preservation of quail blastoderm cells in liquid nitrogen. Br. Poult. Sci. 33: Naito, M., and M. M. Perry Development in culture of the chick embryo from cleavage to hatch. Br. Poult. Sci. 30: Naito, M., A. Tajima, T. Tagami, Y. Yasuda, and T. Kuwana. 1994a. Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J. Reprod. Fertil. 102: Naito, M., A. Tajima, Y. Yasuda, and T. Kuwana. 1994b. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol. Reprod. Dev. 39: Ono, T., and Y. Machida Immunomagnetic purification of viable primordial germ cells of japanese quail (Coturnix japonica). Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 122: Ono, T., and N. Wakasugi Mineral content of quail embryos cultured in mineral-rich and mineral-free conditions. Poult. Sci. 63: Pain, B., M. E. Clark, M. Shen, H. Nakazawa, M. Sakurai, J. Samarut, and R. J. Etches Long-term in vitro culture and characterisation of avian embryonic stem cells with mul-

6 242 PETITTE tiple morphogenetic potentialities. Development 122: Park, T. S., D. K. Jeong, J. N. Kim, G. H. Song, Y. H. Hong, J. M. Lim, and J. Y. Han Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol. Reprod. 68: Perry, M. M Nuclear events from fertilisation to the early cleavage stages in the domestic fowl (Gallus domesticus). J. Anat. 150: Petitte, J. N., M. E. Clark, G. Liu, A. M. Verrinder Gibbins, and R. J. Etches Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108: Petitte, J. N., M. E. Clark, G. Liu, A. M. Verrinder Gibbins, and R. J. Etches Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108: Petitte, J. N., G. Liu, and Z. Yang Avian pluripotent stem cells. Mech. Dev. 121: Reynaud, G The transfer of turkey primordial germ cells to chick embryos by intravascular injection. J. Embryol. Exp. Morphol. 21: Rowlett, K., and K. Simkiss Explanted embryo culture: In vitro and in ovo techniques for the domestic fowl. Br. Poult. Sci. 28: Shaw, D. L., R. S. Carsience, R. J. Etches, and A. M. Verrinder Gibbins The fate of female donor blastodermal cells in male chimeric chickens. Biochem. Cell. Biol. 70: Simkiss, K., K. Rowlett, N. Bumstead, and B. M. Freeman Transfer of primordial germ cell DNA between embryos. Protoplasma 151: Song, Y., S. D Costa, S. L. Pardue, and J. N. Petitte Production of germline chimeric chickens following the administration of a busulfan emulsion. Mol. Reprod. Dev. 70: Speksnijder, G., and R. Ivarie A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs. Poult. Sci. 79: Swift, C. H Origin and early history of primordial germ cells in the chick. Am. J. Physiol. 15: Tajima, A., M. Naito, K. Yasuda, and T. Kuwana Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus). Theriogenology 40: Tajima, A., M. Naito, Y. Yasuda, and T. Kuwana Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gpgcs) in chicken. J. Exp. Zool. 280: Thoraval, P., F. Lasserre, F. Coudert, and G. Dambrine Somatic and germline chicken chimeras obtained from brown and white leghorns by transfer of early blastodermal cells. Poult. Sci. 73: Vick, L., G. Luke, and K. Simkiss Germ-line chimaeras can produce both strains of fowl with high efficiency after partial sterilization. J. Reprod. Fertil. 98: Wei, Q., B. A. Croy, and R. J. Etches Selection of genetically modified chicken blastodermal cells by magnetic-activated cell sorting. Poult. Sci. 80: Wentworth, B. C., H. Tsai, J. H. Hallett, D. S. Gonzales, and G. Rajcic-Spasojevic Manipulation of avian primordial germ cells and gonadal differentiation. Poult. Sci. 68: Wentworth, A. L., and B. C. Wentworth The avian primordial germ cell...an enigma of germline evolution, development and immortality. Avian Poult. Biol. Rev.11: Yasuda, Y., A. Tajima, T. Fujimoto, and T. Kuwana A method to obtain avian germ-line chimaeras using isolated primordial germ cells. J. Reprod. Fertil. 96: Zhao, D. F., and T. Kuwana Purification of avian circulating primordial germ cells by nycodenz density gradient centrifugation. Br. Poult. Sci. 44: Zhu, L., M. C. van de Lavoir, J. Albanese, D. O. Beenhouwer, P. M. Cardarelli, S. Cuison, D. F. Deng, S. Deshpande, J. H. Diamond, L. Green, E. L. Halk, B. S. Heyer, R. M. Kay, A. Kerchner, P. A. Leighton, C. M. Mather, S. L. Morrison, Z. L. Nikolov, D. B. Passmore, A. Pradas-Monne, B. T. Preston, V. S. Rangan, M. X. Shi, M. Srinivasan, S. G. White, P. Winters- Digiacinto, S. Wong, W. Zhou, and R. J. Etches Production of human monoclonal antibody in eggs of chimeric chickens. Nat. Biotechnol. 23: