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This study had two aims. First, we optimized conditions for the lipofection of CHO-K1 cells with transposon vectors. We used flow cytometry to examine the effects of different cell densities, plasmid constructs and lipofection protocols on in vitro transgene expression. Second, we transfected purified chicken primordial germ cells (PGCs) with the most effective lipofection conditions identified in CHO-K1 cells. The use of the CHO-K1 cell model reduced the number of chicken embryos required as PGC donors. CHO-K1 cells (1 x 10 cells/ml or 5 x 10 cells/ml) were transfected with transposon vectors (pCMV-Tol2 with pL-miniTol2-OVA5IFN/pL-miniTol2-OVA5Egfp) via protocol A, B, C or D, the last of which is a control with the nontransposon vector pL-OG-OVAIFNEnh-Egfp. The highest transfection efficiency for transposon vectors was obtained after lipofection of cells with method A (60.0-62.0%), which suggested that this method would be appropriate for transfecting PGCs. Therefore, we performed lipofection of purified PGCs (1 x 10 cells/ml or 5 x 10 cells/ml) using transposon vectors via method A and nontransposon vectors via method D. The most appropriate conditions for introducing transposon vectors into PGCs was lipofection at a density of 5 x 10 cells/ml with method A (transfection efficiency of 67%). We developed an effective protocol for transfecting PGCs using transposon vectors combined with lipofection. The method elucidated in this work will facilitate the production of transgenic chickens in future projects.

Effective Transfection of Chicken Primordial Germ Cells (PGCs) using Transposon Vectors and Lipofection