Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

Takenouchi, Takato; Kitani, Hiroshi; Suzuki, Shunichi; Nakai, Michiko; Fuchimoto, Dai-ichiro; Tsukimoto, Mitsutoshi; Shinkai, Hiroki; Sato, Mitsuru; Uenishi, Hirohide

Isolated primary porcine macrophages were cultured in 60-mm non-tissue culture grade plastic dishes (Sumitomo Bakelite Co., Ltd.). pLVSIN-EF1α neo vectors encoding SV40LT or pTERT were created by Takara Bio, Inc. (Shiga Japan). An artificial Kozak sequence was included before the start codon of each gene to ensure efficient translation (16) . To generate lentiviruses, each of the pLVSIN vectors (2 µg) was co-transfected with packaging vectors (lentiviral high titer packaging mix) (3.25 µg) into Lenti-X 293T cells (Takara Bio, Inc.) (2 × 10 6 cells/60-mm dish) using the TransIT-293 transfection reagent (Takara Bio, Inc.) according to the manufacturer's instructions. Culture supernatants containing lentiviral particles were harvested on day 2, filtered through a membrane filter (pore size: 0.45 µm; Millipore Millex, Millipore Co., Billerica, MA, USA), and used for the porcine macrophage transduction procedures in the presence of 8 µg/ml of Polybrene (Sigma). After several weeks of culturing, proliferative cells appeared, which were then cultured with growth medium containing 800 µg/ml G418 (Thermo Fisher Scientific, Grand Island, NY, USA) to select resistant cells.