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

Abstract

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.

Gamma-irradiation depletes endogenous germ cells and increases donor cell distribution in chimeric chickens

“…Although X-ray irradiation of the germinal crescent in primitive-streak-stage embryos decreases the number of germ cells by up to 75%, this method requires a process for eggshell opening and also causes a number of abnormalities and mortality (Aigegil and Simkiss 1991). Song et al (2005) emulsified busulfan into stage X embryos; however, this method is not appropriate for inducing somatic chimerism at stage X because the long half-life of busulfan (10−18 h) in vivo may inhibit the proliferation of donor cells (Addison and Berenbaum 1971). Reports describing the effects of γ-irradiation have emphasized its role in promoting germline and somatic chimerism, but have generally lacked more detailed data on the change in endogenous PGC numbers after irradiation (Carsience et al 1993;Thoraval et al 1994).…”

“…Kagami et al (1997) removed a section of cells from the central region of the blastoderm to produce chimeric individuals, but the donor cells were not distributed throughout the somatic tissues in the hatchling. Alternatively, the alkylating agent busulfan (1,4-butanediol dimethanesulfonate) may be administrated to stage X embryos to prepare sterilized stage 14−15 embryos (Song et al 2005); however, this method is not appropriate for sterilizing stage X embryos because of subsequent inhibition of donor cell proliferation (Song et al 2005). Other simple methods using X-ray or ultraviolet irradiation have been attempted, but they also resulted in limited success because of a high frequency of abnormalities and the fact that X-ray irradiation produces poor recipient sterilization (Aigegil and Simkiss 1991).…”

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

“…However, in this study, germline transmission rate of the long-term cultured PGC-LCs were low (6%; 3/50). Thus, sterilization of the recipient should be essential for improvement of donorgermline transmission by use of irradiation (Carsience et al, 1993, Atsumi et al, 2009, Macdonald et al, 2010, Nakamura et al, 2012, busulfan treatment (Song et al, 2005, Nakamura et al, 2008. On the other hand, gametic differentiation ability of PGC-LCs might be decreased during long-term culture, this system should be progress so as to improve the ability of gametic differentiation in PGC-LCs.…”

39. The potential of cryopreservation and reproductive technologies for animal genetic resources conservation strategies

“…In the chicken, the primordial germ cells can be specifically harvested. Recently, improvement of the efficiency of producing chimerae with donor genotype germinal cells was achieved by depleting PGC from the recipient embryos using busulfan [44].…”