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 eu karyotic 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 embry os (stage 2), transfected with pCMV beta 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 in creased the transfection efficiency of gPGCs but had no effect on chicken e mbryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfect ion efficiency, Compared with about 17% observed with liposomes. Approximat ely 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipie nt embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to hatching. The exogenous gene was detectable in 100%, 67% and 41% of the 6-day-old (stage 29), 10-day-old (stage 36) recipient embryos and hatch ed chicks gonads, respectively. PCR analysis of DNA from the hatched chicks showed that exogenous lacZ DNA was detected only in the gonad and not the liver and heart. These results indicated that electroporation was a suitabl e means of transfecting avian gPCGs for the goal of producing transgenic po ultry.