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Chimeras and Transgenics: From Greek Mythology to Poultry Breeding Robert J. Etches Department of Animal & Poultry Science University of Guelph Guelph, Ontario, Canada N 1G 2W 1 Introduction In Greek mythology, a chimera is an imaginary fire-breathing animal with the head of a lion, the body of a goat and the tail of a serpent. While the fanciful creation that was killed by the hero Bellepheron as he rode his winged horse Pegasus is not generally contemplated as a research objective by modem biologists, the idea of an animal with parts derived from different sources has broad appeal. In the case of poultry breeding, the appeal of chimeric technology lies in the possibility of combining cells of differing genotypes to obtain chickens that can transmit unique genetic characteristics to their offspring. The unique characteristics may be from cells that have been genetically modified in specific ways using transgenic technology, or from cells that have been cryopreserved for many years. Somatic and germline chimerism Most of the cells in an adult organism are in somatic tissues such as muscle, liver, spleen, pancreas, brain, etc. A much smaller population of cells belongs to the germline, which includes spermatogonia, cells in all stages of spermatogenesis, functional sperm, oocytes and ova. From the point of view of poultry breeding, germline chimerism is much more important that somatic chimerism because only traits that are incorporated into sperm and ova can be transmitted to the next generation. Germline-competent cells The fate of cells during embryonic development can be viewed as an irreversible cascade that begins with a newly fertilized egg. At the one-celled zygote stage, the embryo is described as pluripotential because the lineage of every cell in an adult organism can be traced back to the zygote. As development proceeds, however, cells become committed to specialized functions, such as muscle, liver, spleen, pancreas, brain, etc., and the acquisition of commitment to a specific developmental fate precludes contribution of that cell to alternate developmental fates. Commitment is a gradual process that occurs during the first day of embryonic development in birds. The first day of embryonic development occurs as the egg descends through the reproductive tract and receives its investments of egg white, shell membranes, and shell. During this interval, the embryo divides from a single cell into an embryo containing approximately 50,000 - 60,000 cells in a newly-laid, unincubated egg. At this stage of development, which has been arbitrarily designated as stage X (Eyal-Giladi and Kochav, 1976; Eyal Giladi et al., 1980), the embryo is a radial dome of cells overlying a subgerminal cavity which separates the embryo from the yolk (Fig. 1). The central disk of the embryo is the area pellucida, which is surrounded by the area opaca. Transfer of cells from the area pellucida of stage X embryos to the subgerminal cavity of recipient embryos at the same stage of development produces chimeras (Fig. 2) that are comprised of both donor and recipient cells in somatic tissues and thegermline (Petitte et al., 1990; 1993; Carsience et al, 1993; Thoraval., 1994; Kagami et al., 1995; Kino et al., 1997). The technical details of making chimeras are illustrated in Fig. 3. Mixed-sex chimeras The genome of male chickens includes two copies of the Z chromosome whereas the genome of the female contains one copy of the Z and a W chromosome. There have been many attempts to convert female chickens into males and vice versa to meet the aspirations of the broiler and egg industries, respectively (Etches and Kagami, 1997). All of these attempts have focused on differentiation of the gonad, which begins at about the fifth day of incubation. An alternative strategy to achieve sex reversal is to alter sex determination, which occurs when the egg and sperm unite to form a zygote. This situation can be achieved by injecting male cells into female recipients and vice versa (Fig. 4). When male donor cells are injected into female recipients approximately one half of the chimeras are male and one half are female (Kagami et al., 1995). When female donor cells are injected into male chimeras, however, all of the chimeras are male (Fig. 4). Regardless of the sex of the donor and the recipient, male chimeras derived from male and female blastodermal cells contain genetically female cells in a male environment. This combination provides a powerful experimental model to study sex determination in birds that can be used to determine (1) if genetically female cells can function as spermatogonia in a testis and (2) if genetically male cells can function as ova in females. In brief, when male blastodermal cells are injected into female recipients and a female chimera is produced, the male blastodermal cells are processed normally as ova. When the same combination yields a male chimera, however, genetically female (ZW) spermatogonia can only produce Z-bearing spermatozoa. The W-beating spermatids fail to develop into functional sperm, indicating that there are genes on the Z chromosome that are required for the completion of spermiogenesis. This series of genes may be analogous to those situated on the long arm of the Y chromosome that are essential for complete spermatogenesis in mammals. From the perspective of poultry breeding, the observation that ZZ male cells can become functional female cells indicates that sex reversal from males to females should be possible. The converse may be much more difficult because it is clearly more complicated to produce functional W-bearing sperm. In the future, transgenic technology might be employed to bring together the appropriate genes into parental lines that would yield only male or only female offspring. While this strategy has been applied in plant breeding, its execution on poultry breeding will require more information about sex determination and differentiation in birds. .- i i Creating inbred lines of chickens - While highly inbred lines of chickens have not been used in commercial poultry breeding programs, they have found widespread application in studies of the genetic control of the immune system of chickens (Lamont and Dietert, 1990). In contrast, plant breeding strategies have relied heavily on the creation and hybridization of inbred lines. The difficulty of obtaining inbred lines of chickens has been an impediment to their use in poultry breeding programs, but their development could be greatly facilitated using chimeric technology. Conventional breeding strategies for producing highly inbred lines use 6 to 10 generations of full-sib matings to yield animals that are at least 90% homozygous. However, it is possible to achieve the same level of inbreeding in 3 generations from inter se matings of male and female germline chimeras derived from a single donor embryo (Fig.5). By colonizing the germline of male and female birds with genetically identical cells, it is possible to achieve self-fertilization (Fig.5A). The genome of offspring that result from the first generation of this scheme contains DNA that is derived only from the donor embryo and the only rearrangement of the genome is introduced by segregation. To produce highly inbred populations of chickens, eggs from the offspring of chimeras produced from the same donor embryo would be used as donors for a second cycle of self fertilization. Reiteration of this cycle for 3 generations could provide large populations of highly inbred birds (Fig 5B). Cryopreservation of genetic resources Hundreds of defined breeds, strains and lines of chickens have been developed by research institutes, univerisities, private breeding companies and poultry enthusiasts. Unfortunately, financial constraints prevent the retention of all of these genetic resources as live populations and alternate methods for storing potentially valuable genotypes is required. Semen can be frozen and stored at -196C, and reasonable levels of fertility can be obtained from sperm after thawing and insemination. However, the precise combination of genes cannot be recovered from frozen semen because the line must be outcrossed while inseminating hens with cryopreserved semen. The entire genome can be cryopreserved, however, by freezing blastodermal cells (Fig. 6)i When the line is required in the future, the frozen cells can be thawed and injected into recipient embryos (Fig. 6). Inter se matings of the male and female germline chimeras that are produced yield offspring that are an exact reconstitution of the stock from which blastodermal cells were frozen (Kino et al, 1997). A gene bank has been established at the Univeristy of Guelph that currently holds a number of the lines that were produced by Agriculture and Agri-Food Canada in Ottawa. Expansion of this concept could provide a vast genetic resource from which specific genes or genotypes could be recovered in the future. Creating transgenic chickens via chimeric intermediates The production of transgenic chickens requires modification of the genome of cells that can enter the germline and transmit the genetic modification to the next generation. Since blastodermal cells contribute to the germline, they are excellent candidates for genetic modification. The strategy that is currently being developed at the University of Guelph for the production of transgenic chickens via chimeric intermediates is illustrated in Fig. 7. Briefly, blastodermal cells from donor embryos are dispersed into a single cell suspension and transfected with the gene of interest and a gene conferring antibiotic resistance. Since transfection is usually an inefficient process, cells that have incorporated the genetic modification must be separated from those that remain unaltered. Selection is accomplished by culturing the cells in the presence of the appropriate antibiotic for several days. The selected cells are then transferred to an irradiated recipient embryo to form a transgenic chimera from which founder offspring carrying the transgene are derived. The utility of the transgene would then be evaluated in subsequent breeding programs. Genes that would be inserted into the germline via chimeric intermediates fall into three categories according to their intended use. Genes that might be considered for the poultry breeding industry include those encoding disease resistance and those that might alter the nutritional capabilities of the birds. For example, alleles at the major histocompatability complex might confer partial resistance to Marek's Disease (Bacon and Witter, 1993, 1994a, 1994b) and, therefore, are potentially useful in commercial breeding programs. The incorporation of genes encoding phytase, which would yield chickens that could utilize plant phosphorus more efficiently, would reduce the level of phosphorus in diets and therefore, reduce level of phosphorus in poultry waste. While it may be possible to increase levels of production per se using transgenic technology, it is unlikely that execution Of this strategy will find public acceptance. On the other hand, changes that confer a welfare benefit to the bird, or are perceived as improvements to food safety and quality, are likely to be widely accepted by consumers. The chicken is a widely used experimental model in developmental biology, but its usefulness is currently limited by the lack of ability to create transgenic animals. However, there are many examples of genes that are of interest to developmental biologists who require the ability to make transgenic chickens to execute experimental paradigms that reveal information about gene products and their function. While this use oftransgenic technology lies outside the commercial poultry industry, unexpected benefits to the industry may arise from the rapid increase in knowledge that will be provided by the biomedical research community. A new industry that uses the egg as a repository for proteins encoded by transgenes may become popular if transgenic chickens can be produced routinely. For example, if genes encoding human immunoglobulins (hlg) can be introduced into chickens and if the hlg is deposited in egg yolk, it should be possible to harvest these pharmaceutically important proteins from eggs. The application of transgenic technology to poultry, therefore, has the potential to provide a new set of tools for industrial poultry breeding industry and to spawn new opportunities for poultry breeders that lie outside the traditional domain. 4 References .- Bacon, L.D., Witter (1993). Influence of B-haplotype on the relative efficacy of Marek's disease vaccines of different serotypes. Avian Diseases 37, 53-59 Bacon, L.D., Witter (1994a). B haplotype influence on the relative efficacy of Marek's disease vaccines in commercial chickens. Poultry Science 73, 481-487. Bacon, L.D., Witter (1994b). Serotype specificity of B-haplotype influence on the relative efficacy of Marek's disease vaccines. Avian Diseases 38, 65-71. Carsience, R.S., Clark, M.E., Verrinder Gibbins A.M. and Etches, R.J. (1993). Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117, 669-675 Etches, R.J. (1995). Reproduction in Poultry. CAB International, Wallingford, England. Etches, R.J., Clark, M.E., Toner, A., Liu, G. and Verrinder Gibbins, A.M. (1996). Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture. Molecular Reproduction and Development 45, 291-298 Etches, R.J. and Kagami, H. (1997). Genotypic and phenotypic sex reversal. In: Perspectives in Avian Endocrinology. Edited by S. Harvey and R.J. Etches, Journal of Endocrinology Ltd., Bristol. Eyal-Giladi, H., and S. Kochav, 1976. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stage of the development of the chick. I. General morphology. Dev. Biol. 49: 321-337. Kagami, H, M. E. Clark, A. M. Verrinder Gibbins, and R. J. Etches, 1995. Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline. Mol. Reprod. Dev. 42:379-388. Kino, K., B. Pain, S. P. Liebo, M. Cochran, M. E. Clark and R. J. Etches, 1997. Production of chicken chimeras from injection of frozen-thawed blastodermal cells. Poultry Sci. 76: in press. Kochav, S., M. Ginsburg and H. Eyal-Giladi, 1980. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stage of the development of the chick I. General morphology. Dev. Biol. 79:296-307 Lamont S.J. and Dietert, R.R. (1990). Immunogenetics. In Poultry Breeding and Genetics, edited by R.D. Crawford Elsevier, Amsterdam. Pain, B., Clark, M.E., Nakazawa, H., Sakurai, M. Samarut, J. and Etches, R.J. (1996). Long term culture and characterization of avian embryonic stem cells with multiple morphogenetic capabilities. Development 122, 2339-2348 Petitte, J.N., M. E. Clark, G. Liu, A. M. Verrinder Gibbins and R. J. Etches, 1990. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108:185-189 Petitte, J.N., Brazolot, C.L., Clark, M.E., Liu, G., Verrinder Gibbins, A.M. and Etches, R.J. (1993). Accessing the chicken genome using germline chimeras. In Manipulation of the Avian Genome. Edited by R.J. Etches and A.M. Gibbins. CRC Press, Boca Raton. Thoraval, P., F. Lasserre, F. Coudert and G. Dambrine, 1994. Production of germline chimeras obtained from Brown and White Leghorns by transfer of early blastodermal cells. Poultry Sci. 73:1897-1905. J., 4.4 mat Area opaca i--Area pcHucida_ --I Area#,_ sphcr_ Subgcrminal fluid Blastodermal cells Fig. 1. The structure of an embryo at the time of laying illustrated as a cross section through the embryo perpendicular to the surface of the yolk (from Etches, 1995). Fig. 2. Three chimeras produced by injecting approximately 500 cells from a stage X Barred Plymouth Rock donor embryo into a White Leghorn recipient embryo at the same stage of development. Somatic chimerism is evident from the patches of donor-derived and recipientderived feather pigmentation. Germline chimerism is assessed by mating chimeras to Barred Rocks and examining the down color of the offspring. Black chicks are derived from donor cells that contributed to the germline whereas yellow chicks are derived from recipient contributions to the germline. 7 _ '_'_ .................... •_ Recipient (II) White Leghorn Plymouth Donor Barred (ii) Roc eggs irradiated chimeric chick , / and drilled dispersed '_ rejected into stage X cells recipient chimeric rooster Barred Rock hen Fig. 3. A schematic representation of the procedure for making somatic and germline chimeras. Donor cells are harvested by enzymatic digestion from Barred Plymouth Rock embryos that arehomozygous for the recessive allele at the I locus. Recipient embryos are obtained from White Leghorn eggs that are homozygous for the dominant allele at the I locus and. Within two hourprior to injection of donor cells, development of recipient embryos is arrested for about 24 h by exposing the eggs to approximately 500 rads of), irradiation. Donor cells divide rapidly after injection into the quiescent recipient to yield a chimera that contains substantial contributions from the donor cells. Chimerism in somatic tissues can be assessed from donor-derived down color. Germline chimerism is determined by mating chimeras to Barred Rocks. The proportion of black chicks from this mating is proportional to the donor contribution to the gerrnline. Fig. 4. Schematic diagram for making mixed-sex chimeras. Blastodermal cells from a donor embryo are dispersed and injected into a series of recipient embryos. The sex of the donor embryo is determined by PCR or in situ hybridization of DNA in a sample of donor cells. The sex of the chimera is determined by PCR or in situ hybridization of erythrocytes. Comparison of the sex of the donor embryo and the chimera reveals the sex of the recipient. Injection of male (ZZ) cells into a female (ZW) recipient produces approximately equal numbers of male and female chimeras whereas injection of female cells into male recipients always yields males. ZZ cells in a female chimera are processed in the germline as female cells. ZW cells in a male chimera form spermatogonia and spermatids, but spermiogenesis of W-bearing spermatozoa is blocked. Z-bearing spermatozoa, however, are processed normally into functional spermatozoa. See Kagami et al. (1995) for additional details. (A) X #2 CHIMERIC#3 -'_ CHIMERIC #4 (B) 1 0.9 0.8 g 0.7 _ ,Q_ (t) 0.6 0.5 0.4 _Selfing I _ FullSibs !--&--- Half Sibs r I 0.3 ¢= 0.2 == 0.1 o 0 1 2 3 4 5 Generation Fig. 5. (A) Highly inbred lines of chickens can be produced by dispersing blastodermal cells from a single embryo into a series of recipient embryos. Some of the resulting chimeras will be male and others will be female. Inter se matings between these birds is the equivalent to selfing in plants and produces offspring in which 50% of the genes are homozygous(B). Reiteration of this cycle 4 times produces birds that are 94% homozygous. In comparison to matings between full sibs or half sibs, the level of homozygosity increases much more rapidly when chimeras derived from the same embryo are mated. 10 o Recipient (II) White Leghorn Donor (ii) Barred Plymouth Rock eggs irradiated and drilled "_ dispersed stage X cells thawed cells __'" chimeric chicks injected into _ recipient eggs chimeric rooster cells frozen in liquid _,iiiiii,iiiiiii_ii.m]m!ii i _i-Jiiiiiiiiiiiiii_ _ nitrogen chimeric hen Barred Rock chick Fig. 6. Populations of chickens can be maintained in a cryopreserved state for an indefinite period by freezing blastodermal cells. When the population is required, blastodermal cells are thawed and injected into recipient embryos. To demonstrate this principle, cells from Barred Plymouth Rocks were frozen, stored, thawed and injected into White Leghorn recipients to form chimeras. Inter se matings between the chimeras yielded offspring that were faithful representatives of the Barred Rock population that provided the blastodermal cells for freezing. See Kino et al. (1997) for additional details. 11 Hiiiiiiiii _iiiiiiiiiH !iiiiii!!!_ni.iiiiii_iiii ii!!!_!_iiiiE_ !ii!iiii!i! Gene of interest injected into recipient egg . stage X donor _ modified cells Genetically selected in culture _'" = development of subline homozygous for the genetic modification Fig. 7. A schematic representation of the strategy for producing transgenic chickenS at the University of Guelph. The gene of interest and a gene conferring antibiotic resistance are transfected into dispersed blastodermal cells. The genetically modified cells are then selected by culturing the cells in the presence of the antibiotic. After selection in culture, genetically modified cells are transferred into recipient embryos to form germline chimeras. Donorderived offspring from the chimeras are screened for the presence of the gene of interest. Birds carrying the gene of interest become the founders for a line of chickens that express the desirable trait. 12 • Questions and answers: Dr. A. Tajima Q. Is there any possibility of improving the incorporation rate of donor blastodermal cells into the germ line? A. It may be possible to identify cells within the stage X blastoderm that have the ability to enter the germline. If populations of donor cells that were comprised exclusively germline competent cells was obtained, a higher rate of germline transmission would be anticipated. Dr. M. Delany Q. What is the level of cell death and embryonic death in irradiated receipients? A. We have not estimated cell death. Embryonic mortality after exposure to approximately 500 rads is about 20% (see Carsience et al., 1993). Q. What is the mutation rate in recipient embryos and is that important for developoment of the chimeras and their offspring? A. We have reared irradiated recipients and examined their offspring for evidence of mutation. Fertility and hatchability of irradiated recipients is normal and no morphological abnormalities were observed among the offspring. From these data we have concluded that there are no major effects of the low dose of irradiation. However, the recipient is only a vehicle for transferring cells that have been genetically modified from a culture vessel into the germline of a reproductively active bird that can produce transgenic offspring. Recipient embryos do not contribute to the transgenic gene pool and, therefore, any potentially detrimental effects of irradiation would be excluded from the subsequent development of a line of birds expressing the transgene. Q. Have you tried to make chimeras with turkey embryos? A. No, we have not. Dr. Steve Stice Q. For how long can you maintain blastodermal cells in culture? 13 A. We have been able to make somatic and germline chimeras from cells that have been in culture for up to 7 days, but have kept the cells in vitro for much longer periods. See Etches et al. (1996) and Pain et al. (1996) for additional details. Q. Is this long enough to get selection of the gene for making transgenic animals and possibly gene knockouts? A. Seven days provides sufficient time to obtain significant enrichment of the population of transfected cells and may provide sufficient time to make a knockout. This is an area that we are actively pursuing at the moment. Dr. Douglas Rhoads Q. Have you tried sex reversal of the chimera based on recipient genotype? A. All combinations of male and females as donor and recipients have been investigated but the combinations have been identified retrospectively. See Kagami et al. (1995) for additional details. 14