Analysis of Transgene Intergration Efficiencies into Porcine Fetal Fibroblast Using Different Transfection Methods and Twice Drug Selection.

Abstract

Transgenic animals can be produced by nuclear transfer with genetically modified somatic cells in which the procedure of transgene transfection is required. The objective of this study was to compare transfection efficiencies of electroporation (Microporater [Seo Ryn Bio, Korea]) and liposome reagents (FuGENE 6, FuGENE HD [Roche, USA], Welfect-ex, Welfect-Q, Welfect-M [Welgene, Korea]) into pig fetal fibroblast cells using transgene vector containing human tPA cDNA fused to β-casein promoter sequence and neomycin-resistant gene (Neor) driven by PGK promoter and to determine the proportion of cells containing transgene in neomycin-resistant colonies. The linearized transgene vector DNA was transfected into pig fetal fibroblasts with electroporation or different transfection reagents according to manufacturer's procedures. Pig fetal fibroblast cells were selected following exposure to 400 μg/ml G418 for 14 days. Cell colonies surviving G418 selection were picked by cloning cylinder (Sigma, USA) and assayed by PCR amplification with tPA-specific primers. Also, single cell PCR analysis of growing colonies resistant to G418 selection was carried out to evaluate the percentage of cells containing transgene in G418-resistant colonies. Among transfection methods tested in this experiment, electroproation exhibited significantly higher transfection efficiency compared with liposome reagents (50% with microporater vs 20% with liposomes). When cells of G418-resistant colonies were performed by single-cell PCR, 60 to 100% of cells in each colony were identified to contain transgene regardless of transfection methods. The colonies carrying 60 to 80 % transgene-positive cells were cultured again with 400 μg/ml G418 for 14 days, and cell colonies surviving G418 selection were selected and assayed by PCR. Transfection frequencies in theses colonies for tPA gene were 95% to 100%. The present results suggested that electroproation may increase transfection efficiency of DNA into pig fetal fibroblast cells compared with liposome methods and twice drug-selection can improve transfection rate.