Long Term in vitro Culture of Chicken Primordial Germ Cells Isolated from Embryonic Blood and Incorporation into Germline of Recipient Embryo

Abstract

The present study was carried out to develop a long-term culture system for chicken primordial germ cells (PGCs). PGCs were obtained from the blood of .-day incubated embryos. GFP gene was transferred into the collected PGCs and then cultured on feeder cells derived from the gonads of-day incubated embryos. The GFP-positive cells attached loosely to the feeder cells, and their morphology varied from round to fibroblast-like in shape. They proliferated slowly and occasionally formed colonies. The PGCs cultured for days were transferred into the bloodstream of recipient embryos, and we examined their incorporation into the germline of chimaeric chickens. Test mating was carried out, and one germline chimaeric chicken was detected out of putative chimaeric chickens. That one chicken was generated by transferring-day cultured PGCs and it produced one donor-derived o spring out of examined. Thus, a small percentage of the cultured PGCs retained the ability to migrate to the germinal ridges, thus giving rise to functional gametes, although most of the cultured PGCs di erentiated during the culture period. In conclusion, a germline chimaeric chicken was generated by transferring PGCs cultured for about months; the culture system for PGCs developed in the present study will contribute to the germline manipulation in chickens. : chicken, culture, embryo, germline chimaera, primordial germ cell the risk of a cross-transfer of animal pathogens from other animal cells. It is, therefore, recommended to use chicken cells as feeder cells for culturing chicken PGCs. Chicken PGCs isolated from embryonic blood could be

Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

“…PGCs cultured on feeder cells proliferated slowly, occasionally forming cell colonies. Although they retained the characteristics of germline cells, it was difficult to maintain an undifferentiated state during the culture period similar to those from our previous results (Naito et al, 2010). bFGF plays an important role in maintaining the undifferentiated state of cultured PGCs (van de Lavoir et al, 2006;Choi et al, 2010;Macdonald et al, 2010), and also the foetal bovine serum also affected the proliferation and maintenance of an undifferentiated state of cultured PGCs (Macdonald et al, 2010).…”

“…Freshly collected PGCs usually migrate to the recipient gonads by transferring them into the stage X blastoderm (Naito et al, 2004) or bloodstream (Yasuda et al, 1992;Tajima et al, 1993;Naito et al, 1994aNaito et al, , 1994bNaito et al, , 2010. On the other hand, when cultured PGCs were transferred to the coelomic epithelium of recipient embryos, they were able to enter the gonads, as in our previous report (Naito et al, 2011).…”

“…Although the former method is effective for introducing exogenous genes into chickens, it has disadvantages: it restricts transgene size and poses safety problems. Therefore, manipulation of germline cells is currently considered to be the more useful method for gene transfer into chickens (Naito et al, 2010). Since primordial germ cells (PGCs) are progenitor cells of ova and spermatozoa, germline chimaeric chickens can be produced by transfer of PGCs between embryos (Tajima et al, 1993;Naito et al, 1994aNaito et al, , 1994bNaito et al, , 1998Naito et al, , 1999).…”

“…In our previous study, PGCs isolated from the blood of 2.5-day incubated embryos were cultured on feeder cells derived from the gonads of 7-day incubated chicken embryos (Naito et al, 2010). The cultured PGCs proliferated even after transfecting GFP gene, and produced a germline chimaeric chicken after transferring them into the bloodstream of recipient embryos.…”

Isolation, Cultivation and Characterization of Quail Primordial Germ Cells

“…The production of transgenic and chimeric individuals based on primordial germ cells (PGC) is recognized as one of the promising areas of modern biotechnology, which are discussed as an alternative to traditional methods of genome selection and modification [1][2][3][4][5]. The use of this type of cells in selection and biotechnological programs opens wide opportunities for directed genome modification and the reconstruction of valuable breeds and lines preserved in cryobank conditions [6][7][8][9][10][11][12].…”