SV40 Large T Antigen Lentivirus (EF1α)

Primary canine corneal epithelial cells (CCEC) are prone to senescence and cell proliferation is limited. Experimental sampling requires a large number of animals, which poses animal welfare issues and cannot maintain the stability of cells for in vitro analysis. In this study, CCEC was isolated and purified by trypsin and dispase II enzymatic hydrolysis, and then immortalized by transfecting a lentiviral vector expressing simian vacuolating virus 40 large T (SV40T). An immortalized canine corneal epithelial cell line (CCEC-SV40T) was established by serial passage and monoclonal selection. After 40 generations of continuous passage, CCEC-SV40T cells grew in a cobblestone shape, similar to CCEC, and the SV40T gene and cytokeratin 12 could be detected in each generation. CCEC-SV40T cells have a stronger proliferation ability than CCECs, and maintain a diploid karyotype and serum dependence like CCECs. After CCEC-SV40T cells were infected with S. pseudintermedius, the mRNA expression of NLRP3, Caspase-1 and proinflammatory factors IL-1β, IL-6, IL-8, and TNF-α was upregulated, the protein expression of MyD88 and NLRP3 was upregulated, and the phosphorylation of Iκbα and p65 was upregulated. Therefore, the CCEC-SV40T cell line was successfully established and can be used for in vitro studies such as corneal disease research or drug screening.

Here, the researchers used the SV40 Large T Antigen Lentivirus to immortalize canine corneal epithelial cells. PCR results showed that the SV40T gene was not expressed in CCECs, but could be detected in 293T cells and CCEC-SV40T cells of different generations (Figure 1A). The above results indicate that the SV40T gene was successfully transfected into canine corneal epithelial cells and was stably expressed with cell passage. During the passage process, the morphology of CCEC-SV40T cells did not change (Figure 1B). The proliferation characteristics of CCEC-SV40T cells were evaluated by cell growth curve and cell cycle, and the proliferation rate of CCEC-SV40T cells was significantly faster than that of CCECs (Figure 1C). Cell cycle detection showed that the proportion of cells in the S phase in CCEC-SV40T cells was greater than that in CCECs (Figure 1D). Cell immunofluorescence results showed that CCEC-SV40T cells of different generations expressed cytokeratin 12 (red fluorescence; Figure 1E). Karyotype analysis of CCEC-SV40T cells showed that both CCEC and CCEC-SV40T cells maintained a diploid karyotype without obvious chromosomal abnormalities (2n = 78; Figure 1F).

Figure 1. Characteristics of the CCEC-SV40T line. (Guo L, et al., 2022)