GENE TRANSFECTION OF MOUSE PRIMORDIAL GERM-CELLS IN-VITRO AND ANALYSIS OF THEIR SURVIVAL AND GROWTH-CONTROL

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pS V-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transi ently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days post coitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shap e and exhibit growth arrest during a few days of culture, and these ro unded PGCs quickly disappear from the culture. We found that the trans fection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molec ular-weight protein (E1B 19K) significantly promoted the survival of P GCs and retarded the disappearance of rounded PGCs from the culture sy stem. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in c ulture. (C) 1997 Academic Press