Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.     

Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus)

Blood was collected from Stage 13 to 14 (1) chick embryos. Primordial germ cells (PGCs) were separated from blood cells by Ficoll density gradient centrifugation. One hundred Rhode Island Red PGCs per embryo were transferred to the blood stream of Stage 14 to 15 White Leghorn embryos. Also, one hundred White Leghorn PGCs per embryo were transferred to the blood stream of Stage 14 to 15 Rhode Island Red embryos. Hatched male and female chicks were raised until sexual maturity, and progeny tests were performed by mating these PGC recipients with Rhode Island Red chickens of the opposite sex. Chicks apparently derived from the transferred PGCs, based on the feather color of the chicks, were produced from all 4 possible mating combinations. The present results indicate that the germ line of PGC recipient chickens consists of 2 distinct populations of germ cells.     

Study on the variation of precipitin reactions in normal sera of chickens

Breed distribution of precipitin reactants (of types A and B) was encountered in: Brahma and Columbian Plymouth Rock - high strength and frequencies; White Cornish, Dark Cornish, Minorca, Barred Plymouth Rock, New Hampshire, Rhode Island Red, Catalana and Cubalaya - low to medium strength and frequencies; Australorp, White Leghorn, Polish, Sexline - no reactions. Cocks exhibited only type A reactants. Both A and B substances were detected in egg yolks. Type A substances were encountered in sera of newly hatched chicks. Their decay was slow - the mean persistence time being about 15 days. Only two young chicks among 160 tested exhibited the reactants of type B. Hormonal treatments of White Leghorn and Rhode Island Red cocks with testosterone propionate, progesterone, oestradiol benzoate or oestradiol benzoate along with progesterone failed to induce the synthesis of precipitin reactants.     

Expression of mRNA for 3HADH in manipulated embryos to produce germline chimeric chickens

Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12–15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.     

Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.     

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Germline chimeric chickens from FACS‐sorted donor cells

Germline chimeric chickens can be constructed by injecting donor chicken blastodermal cells (CBCs) into recipient embryos and incubating to hatch. Transgenic chickens can be produced through chimeric intermediates if the donor cells are genetically manipulated; the chance of producing a transgenic chimera would be increased by enriching the donor population in transfected cells. To demonstrate that donor CBCs can be sorted according to the expression of a foreign gene, CBCs in suspension were subjected to transfection with plasmid DNA encoding bacterial β‐galactosidase (β‐gal). Following an overnight incubation, the CBCs were loaded with 5‐dodecanoylaminofluorescein di‐β‐D‐galactopyranoside (C₁₂FDG), which is fluorescent after cleavage by β‐gal. The treated cells were subjected to fluorescence activated cell sorting (FACS) to give “positive” (fluorescent) and “negative” (non‐fluorescent) populations. Almost 100% of the “positive” population showed β‐gal activity. “Positive” cells were cultured on mouse SNL 76/7 fibroblast feeder cells and formed colonies, most of which still stained positively for β‐gal activity after three days. FACS‐sorted cells of Barred Plymouth Rock origin were injected into recipient White Leghorn embryos, resulting in chimeric embryos. Of the 298 embryos injected with sorted cells, 23 (8%; 18 injected with “positive cells, five with “negative”) survived to rearing. Somatic chimerism was seen in 12 of 18 (67%) “positive” and three of five (60%) “negative” birds with the proportion of black pigmentation averaging 19% overall. Twenty birds reached sexual maturity, of which 12 (60%) were somatically chimeric; seven (35%) of these produced donor‐derived chicks. Donor CBCs can, therefore, be sorted by FACS according to the expression of a selectable marker gene without impairing their ability to contribute to germline chimeras; this procedure could be incorporated into a practicable method by which to increase the chances of producing a transgenic chicken. Mol. Reprod. Dev. 52:33–42, 1999. © 1999 Wiley‐Liss, Inc.     

Detection and characterization of primordial germ cells in pheasant (Phasianus colchicus) embryos

The developmental similarity between the chicken and pheasant (Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff's solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62-72 h or from gonads from embryos incubated for 156-216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater (P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66-72 h (31.4 versus 5.6-16.2 microL(-1)). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193-2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50-0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.     

Inter embryonic (homo- or hetero-sexual) transfer of primordial germ cells between chicken embryos

Chicken primordial germ cells (PGCs) collected from thecirculating blood in embryonic vessels at stage 13–15 were inter-embryonically, homo- or hetero-sexually,transferred to the blood vessels of recipient embryosat the same stage of development. Approximately 30%of the embryos treated with hetero-sexual transfer of PGCs had abnormal gonads, showing ovotestis likeorgans. In this case, some of these reversed gonadswere considered to be dependent upon the ratio of thenumber of PGCs from donor to recipient embryos. Oneof the treated embryos possessed completely reversedorgans. Therefore, the introduction of exogenousembryonic vessels was thought to be also useful forproducing transgened gonads.     

Xenogenic oogenesis of chicken (Gallus domesticus) female primordial germ cells in germline chimeric quail (Coturnix japonica) ovary

In present study, chicken primordial germ cells (PGCs) were transferred into quail embryos to investigate the development of these germ cells in quail ovary. Briefly, 2 microl of chicken embryonic blood (stage 14) or about 100 purified circulating PGCs were transferred into quail embryo. Contribution of chicken PGCs were detected in gonads of chimeric quail embryos (stage 28) by immunocytochemical staining of cell surface antigen SSEA-1, and by in situ hybridization (ISH) with female chicken specific DNA probe. As a result, 52.0+/-43.2 (n=18) and 42.7+/-27.3 (n=17) chicken PGCs were found in the gonads of chimeric quail embryo that was injected with chicken embryonic blood (stage 14) and about 100 purified circulating PGCs, respectively. Furthermore, the ovaries of 81.8% (9/11) 12 days post incubation (dpi) chimeric quail embryos were observed with a mean of 457.6+/-237.1 female chicken PGCs-derived oogonia scattered in ovarian cortex area. In 9 out of 12 newly hatched and one week old chimeric quail chicks, on average of 2883.0+/-1924.1 primary oocytes and 3 follicles derived from chicken PGCs were found, respectively. The present results suggest that chicken female PGCs are able to migrate, colonize, proliferate and differentiate into oogonia, primary oocytes in chimeric quail ovary.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

Improved Transfection Efficiency of Chicken Gonadal Primordial Germ Cells for the Production of Transgenic Poultry

Electroporation is a common method of DNA transfection for many types of eukaryotic cells, but has not been attempted in avian primordial germ cells (PGCs). DNA uptake in chicken primordial germ cells (PGCs) was tested using electroporation with and without dimethyl sulfoxide (DMSO). Gonadal tissue and chicken embryonic fibroblasts (CEFs) were isolated from 6-day-old embryos (stage 29), transfected with pCMV carrying the bacterial lacZ gene, and cultured for 24 h. Gonadal primordial germ cells (gPGCs) were purified from culture using a Ficoll gradient. The addition of DMSO significantly increased the transfection efficiency of gPGCs but had no effect on chicken embryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfection efficiency, compared with about 17% observed with liposomes. Approximately 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipient embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to ...     

Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species

Background

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.

Methodology/Principal Findings

Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.

Conclusion

This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.     

Strain preference in donor and recipient for production of W-bearing sperm in mixed-sex germline chimeric chickens

To elucidate the strain preference in donor and recipient for the production of W-bearing sperm, mixed-sex germline chimeric chickens were produced. The combination of donor and recipient was White Leghorn (WL) and Barred Plymouth Rock (BPR), and vice versa. Four sets of mixed-sex chimeras that had the male phenotype at sexual maturity were subjected to analysis: group 1, a female WL donor and a male BPR recipient; group 2, a male WL donor and a female BPR recipient; group 3, a female BPR donor and a male WL recipient; group 4, a male BPR donor and a female WL recipient. The mean number of W-bearing sperm detected by in situ hybridization among 10000 sperm observed was 135, 158, 26 and 71 in groups 1, 2, 3 and 4, respectively. The number in group 1 was significantly higher than that of group 3 (P<0.05). And the number in group 2 was significantly higher than those of groups 3 and 4 (P<0.05). It is suggested that the combination of a WL donor and a BPR recipient produced W-bearing sperm more efficiently than the reverse combination.     

Heterotopic transplantation of testes in newly hatched chickens and subsequent production of offspring via intramagnal insemination

Transplantation of testicular tissue onto the back of immunodeficient nude mice provides a tool to examine testicular development and preserve fertility in mammals. There is no immunodeficient model in birds, but we recently transplanted ovarian tissue between newly hatched chicks from two lines of chickens and produced donor-derived offspring, showing that experimental transplantation is possible in newly hatched chicks. In the present study testicular tissue from newly hatched Barred Plymouth Rock (BPR) chicks was transplanted under the skin of the back, under the skin of the abdomen, or in the abdomen of White Leghorn chicks that had been surgically castrated and immunocompromised. Recipient birds were killed at 10 mo of age. Transplanted tissue was observed in one of five hosts receiving tissue under the skin of the back, two of five hosts receiving tissue under the skin of the abdomen, and three of five chicks with grafts inside the abdominal cavity. In recipients with no regeneration of host testes, testicular transplants grew to the size of normal testes, and histologic analysis showed active spermatogenesis. Subsequent collection of sperm from two successful transplants and surgical insemination of the sperm into the magna of the oviducts of BPR hens resulted in the production of 24 donor-derived chicks. These results demonstrate that the combination of testicular tissue transplantation with intramagnal insemination can produce viable, normal chicks, which could provide a simple approach for the recuperation of live offspring in avian species.     

Molecular and biological aspects of early germ cell development in interspecies hybrids between chickens and pheasants

Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications.     

Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos

In the avian species, germline chimera production could be possible by transfer of donor germ cells into the blood vessel of recipient embryos. This study was conducted to establish an efficient transfer system of chicken gonadal primordial germ cells (gPGCs) for producing the chimeras having a high capacity of germline transmission. Gonadal PGCs retrieved from 5.5-day-old embryos (stage 28) of Korean Ogol chicken (KOC with i/i gene) were transferred into the dorsal aorta of 2.5-day-old embryos (stage 17) of White Leghorn chicken (WL with I/I gene). Prospective evaluations of whether culture duration (0, 5, or 10 days) and subsequent Ficoll separation of gPGCs before transfer affected chimera production and germline transmission in the chimeras were made while retrospective analysis was conducted for examining the effect of chimera sexuality. A testcross analysis by artificial insemination of presumptive chimeras with adult KOC was performed for evaluating each treatment effect. First, comparison was made for evaluating whether experimental treatments could improve chimera production, but none of the treatments were significantly (P = 0.6831) influenced (5.1%-14.4%). Second, it was determined whether each treatment could enhance germline transmission in produced chimeras. More (P < 0.0001) progenies with black feathers (i/i) were produced in the germline chimeras derived from the transfer of 10-day-cultured gPGCs than from the transfer of 0- or 5-day-cultured gPGCs (0.6%-7.8% vs. 10.7%-49.7%). Ficoll separation was negatively affected (P < 0.0001), whereas there was no effect in chimera sexuality (P = 0.6011). In conclusion, improved germline transmission of more than a 45% transmission rate was found in chicken chimeras produced by transfer of 10-day-cultured gPGCs being separated without Ficoll treatment.     

PRODUCTION OF GERMLINE CHIMERIC CHICKENS BY TRANSFER OF CULTURED PRIMORDIAL GERM CELLS

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.