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

Since isolated primary PKM that have become attached to plastic dishes cease to proliferate under culture conditions that do not include growth promoters (e.g., granulocyte-macrophage colony-stimulating factor or colony-stimulating factor 1), it is easy to determine whether a cell has acquired the ability to proliferate indefinitely. Based on the findings of a previous study (9) , primary PKM were transfected with the pSV3-neo plasmid (ATCC Number: 37150) using the Lipofectamine (Thermo Fisher Scientific) or FuGENE HD (Promega) transfection reagent. However, we were unable to obtain an immortalized cell line that retained the characteristics of macrophages from these transfected cells. This was probably because the primary PKM were sensitive to the toxic effects of these transfection reagents. Thus, we decided to use lentiviral vectors to transfer immortalizing genes into the primary PKM. Initially, the cells were infected with lentiviral particles carrying the gene for SV40LT. However, no immortalized cells were generated from the SV40LT-transfected PKM. Next, the cells were transfected with lentiviral particles carrying the SV40LT gene in combination with lentiviral particles carrying the pTERT gene. After several weeks of culturing, proliferative cells appeared in the plastic dishes and were routinely passaged. The expression of both SV40LT and pTERT mRNA was detected in these cells by Q-PCR analyses (data not shown), suggesting that the co-expression of SV40LT and pTERT was effective at immortalizing the primary PKM. Thus, we successfully produced an IPKM cell line.