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Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

1. A series of experiments were conducted to examine the developmental potential of cryopreserved gonadal germ cells (GGCs) recovered from both males and females on embryo day 7 (7 d-GGCs) using the PBS(-) method. Germline chimeras were produced by transferring 200 frozen/unfrozen 7 d-GGCs recovered from female/male Rhode Island Red (RIR) embryos into the dorsal aorta of 2-day-old female and male white leghorn (WL) embryos.2. Germ-cell recipient embryos were hatched and raised to sexual maturity and progeny testing was conducted by mating with RIR of the opposite sex. Brown-feathered progeny chicks were hatched in all eight possible progeny testing combinations, except for male GGC recipients produced by transferring female GGCs. Furthermore, brown-feathered progeny chicks were hatched when frozen-thawed sperm from male germline chimeras, produced by transferring unfrozen 7d-GGCs, were inseminated in normal female RIR and female WL germline chimeras.3. The results indicated that cryopreserved female/male GGCs from 7-day-old chick embryos, recovered using the PBS(-) method, were fully capable of developing into normal spermatozoa and ova in the gonad of recipient embryos under appropriate GGC donor/recipient combinations.

Germ cell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germ cell transplantation systems have been developed, including sperm, oocyte, or germline stem cell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germ cell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.

Birds obtained by embryo engineering are used to study embryo development and to produce transgenic birds. As this method of producing birds still generate strong emotions of the public opinion head ornaments, testes and semen characteristics of sex chimera roosters were examined to check whether they differ from chickens obtained by non-manipulated methods. Measurements of head ornaments, testes and semen were correlated with each other. Semen quality factor (SQF) was calculated, as well as the level of fluctuating asymmetry (FA) of bilateral traits (wattles and testes). Positive correlation was found for comb width and wattle length and comb thickness and sperm concentration. Semen characteristics and FA did not exceed the level encounter in other chicken lines. Results obtained indicate that germline chimeras are similar in appearance of secondary sexual traits, and semen and testes characteristics to chickens produced in non-manipulated way., (© 2018 Blackwell Verlag GmbH.)

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enablingchicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containingPGCs obtained from 2.5-day-old chicken embryos accordingto the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced duringculture in the basic medium containingeither basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defininga culture condition enablingPGCLCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

ABSTRACT The development of chicken embryo culture techniques, from single-cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro . Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single-cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non-viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary. [ABSTRACT FROM AUTHOR]