Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Background: Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which were isolated from pig respiratory tract and were mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan.Results: In this study, we carefully isolated and cultured primary PTECs. Besides, we constructed immortalized PTECs by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT). Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR. hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, using this cell line, the lack of transformed and grown tumors in nude mice indicated that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas and were suitable as a cell adhesion model.Conclusions: In summary, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in the infection study of various porcine respiratory pathogens.