409 EFFICIENCY OF CO-TRANSFECTION OF TRANSGENE AND Neor GENE INTO GOAT AND PIG FETAL FIBROBLASTS

Transgenic animals can be generated by nuclear transfer with genetically modified somatic cells in which the essential procedure of transgene transfection is required. Most transgene vectors are constructed to contain transgene and drug-resistant genes to enrich for somatic cells in which transgene integration has occurred. However, construction of transgene vectors along with drug-resistant genes may not be easy, due to inappropriate restriction sites. Therefore, in this study, two separate constructs, human tPA cDNA fused to β-casein promoter sequence as a transgene vector and neomycin-resistant gene (Neor) driven by PGK promoter as a drug-selectable gene, were co-transfected into pig and goat fetal fibroblast cells to estimate the efficiency of transgene transfection following G418 selection. First, goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) were tested for G418 resistance with different concentrations of G418. The pertinent concentrations of G418 were 800 µg mL−1 for GFF and 200 µg mL−1 for PFF. The linearized tPA vector and Neor gene vector were co-transfected into goat fetal fibroblasts and pig fetal fibroblasts with FuGENE6 transfection reagent (Roche Diagnostics, Mannheim, Germany). The cells were selected following exposure of 800 µg mL−1 and 200 µg mL−1 G418 for GFF and PFF, respectively, for 14 days. Cell colonies surviving G418 selection were assayed by PCR amplification with tPA-specific primers. Initially 2 × 106 GFF and PFF were transfected. Resistant colonies were counted and transferred to 24-well plates for expansion and PCR analysis. The results of co-transfection experiments are summarized in Table 1. The transfection of 2 × 106 GFF and PFF yielded an estimated 96 and 93 colonies, respectively, which survived as the G418 selection. However, 54 colonies of GFF and 39 colonies of PFF proliferated during expansion and were subjected to PCR analysis. Twenty-three and 5 of these colonies were identified to contain tPA transgene in GFF and PFF colonies, respectively. Transfection frequencies for tPA gene were 42.6% and 12.8% in GFF and PFF, respectively. These results suggest that co-transfection of transgene vector with Neor gene can be an alternative method for transfection of transgenes into fetal fibroblast cells. Table 1. Transfection efficiency of goat fetal fibroblasts (GFF) and pig fetal fibroblasts (PFF) following co-transfection of tPA gene and Neor gene