| Cell culture technology is an important research tool in cell biology, virus isolation and propagation, and vaccine production. Cell lines have unlimited propagation capability in vitro, including spontaneous immortalized cell lines and cancer or viral gene-transformed cell lines. The spontaneous immortalized cells are normal cells, but their occurrence is very rare. The transformed cell lines are relatively easy to establish, but have biosafety concerns due to the presence antibiotic resistant and/or viral genes. Therefore, the immortalized cell lines without antibiotic resistant and/or viral genes have extensive applications in cell biology and vaccine development.Telomere is a special structure at the ends of eukaryotic chromosomes and plays important roles in cell propagation and aging. The telomere DNA is replicated by telomerase, which consists of telomere RNA, telomere-associated proteins and telomerase reverse transcriptase (TERT). Previous studies have shown that TERT gene transfer can be used to establish immortalized cell lines. The main gene transfer methods include plasmid vector transfection and viral vector transduction. The former method has a low efficiency and needs antibiotic resistant selection for stably transfected cell clones, while the later method is relatively easy with biosafety concerns. Transposons are mobile sequences within cell genomes and have been used as gene transfer vectors. Due to their higher transfection efficiency and ability to mediate foreign gene integration into host cells, no antibiotic selection is needed for generation of stably transfected cell lines.By combining transposon-mediated gene transfer with TERT-mediated cell immortalization, in this study a sleeping beauty (SB) transposon vector, named as pTEG, was constructed by using porcine elongation factor1a (EF-1a) promoter and bovine growth hormone polyA signal. By using green fluorescent protein (GFP) gene as a reporter, cell transfection assay showed that porcine EF-la promoter had a strong transcription activity, which was comparable to that of the early promoter of human cytomegalovirus. Co-transfection study with the transposon vector and transposase vector showed that the transposon system could mediate the reporter gene integration into porcine cells.Then, we cloned porcine TERT cDNA and inserted it into the transposon vector pTEG. Primary porcine fibroblast cells (PFCs) were co-transfected with the recombinant vector pTEG-TERT and the transposase vector pSB16. After brief cultivation, monoclonal cell clones were obtained, from which two cell clones with the TERT expression cassette but not antibiotic resistant gene were selected. The immortalized cell clones had been passed in vitro for more than50generations without losing propagation capability. Morphological study showed that the immortalized cells had a typical fibroblast cell morphology and cell growth curve. Compared to the primary PFCs, the immortalized cells had a shorter doubling time and higher cloning efficiency. Cell cycle experiment showed that the immortalized PFCs had a similar cell cycle but significantly higher cell number in the S phase compared to the primary cells. Cell marker detection showed that the immortalized PFCs expressed the biomarkers of primary fibroblasts. Cell transplantation study showed that the immortalized PFCs could not form the tumor cell colonies in mice. Further studies showed that the immortalized PFCs remained the susceptibility to pseudorabies virus infection without mycoplasma contamination. These experimental data suggest that the transposon vector pTEG-TERT generated in this study can be used for porcine immortalization and the PFC cell line generated is useful for pig virus isolation and vaccine development. |
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