Establishment of porcine transgenic embryonic germ cell lines expressing enhanced green fluorescent protein.

The purpose of this study was to isolate porcine embryonic germ (EG) cells and establish transgenic EG cell lines. Plasmid DNA was the enhanced green fluorescent protein (EGFP) vector. Porcine EG cells in rapid proliferation (4th to 9th passage) were transfected with LipofecTamine 2000 and TransFast reagents. Porcine EG cells transfected with a complex of 1 microg of DNA and 2 microL of LipofecTamine 2000 reagent yielded four EG-EGFP cell lines, which emitted bright green fluorescence. EG-EGFP cells cultured for more than 2 weeks without passage gave rise to various differentiated phenotypes. In addition, to determine the degree to which EG cells become integrated into the inner cell mass of host embryos, 135 embryos were injected with porcine EG-EGFP cells; 110 embryos survived and developed into blastocysts (81.5%). Eighty-four chimeric embryos contained fluorescent cells after culture; 49 blastocysts contained EG-EGFP cells in the inner cell mass. Our results suggested that the chimeric rate would not be improved via using different stages of embryos for injection.