Retrovirus-mediated in vitro gene transfer into chicken male germ line cells

Abstract

Chicken testicular cells, including spermatogonia, transplanted into the testes of recipient cockerels sterilized by repeated g-irradiation repopulate the seminiferous epithelium and resume the exogenous spermatogenesis. This procedure could be used to introduce genetic modifications into the male germ line and generate transgenic chickens. In this study, we present a successful retroviral infection of chicken testicular cells and consequent transduction of the retroviral vector into the sperm of recipient cockerels. A vesicular stomatitis virus glycoprotein G-pseudotyped recombinant retroviral vector, carrying the enhanced green fluorescent protein reporter gene was applied to the short-term culture of dispersed testicular cells. The efficiency of infection and the viability of infected cells were analyzed by flow cytometry. No significant CpG methylation was detected in the infected testicular cells, suggesting that epigenetic silencing events do not play a role at this stage of germ line development. After transplantation into sterilized recipient cockerels, these retrovirus-infected testicular cells restored exogenous spermatogenesis within 9 weeks with approximately the same efficiency as non-infected cells. Transduction of the reporter gene encoding the green fluorescent protein was detected in the sperms of recipient cockerels with restored spermatogenesis. Our data demonstrate that, similarly as in mouse and rat, the transplantation of retrovirus-infected spermatogonia provides an efficient system to introduce genes into the chicken male germ line. Reproduction (2007) 134 445-453

Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses

“…The replication-defective MLV-based vector pLG and its propagation from a molecular clone were described previously (44). Infection of 293T and TE671 cells was done in the same way as that for the PERV 14/220.…”

The reversible developmental unipotency of germ cells in chicken

“…The other possibility is that PGCs freshly isolated and transferred into adult testis could not overcome the developmental gap for (de)differentiation and maturation or actually, the transplanted PGCs were a mixed population retrieved from male and female embryos which probably affected germ cell differentiation in the recipient testis. Although both the PGC/EGC-to-embryo and SSC/GSC-to-testis germline transmission systems can generate germline chimeras or transgenic animals (van de Lavoir et al 2006, Kalina et al 2007, Shin et al 2008, Han 2009), each possesses different merits. The classical embryo-mediated system is more practical than the testis-mediated system and detailed information is available for the chicken.…”

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

“…This technique has now been used in a number of animal species, including domestic mammals [2], fish [3], and chicken [4,5]. Donor germ cells repopulate the spermiogenic epithelium in recipient's testis and produce fertile donor-derived sperm, such that the recipient male can distribute the genetic material of the germ cell donor.…”