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

Abstract

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.

Immune Escape Adaptive Mutations in Hemagglutinin Are Responsible for the Antigenic Drift of Eurasian Avian-Like H1N1 Swine Influenza Viruses

“…To determine whether both escape mutants maintained a level of replicative ability similar to that of the parent HeN11 virus, we examined virus growth kinetics in Madin-Darby canine kidney (MDCK) cells and human telomerase reverse transcriptase-immortalized porcine tracheal epithelial cells (hTERT-PTECs), which were prepared by transfecting primary PTECs with hTERT ( 30 ). Compared with that of the parent HeN11 virus, the replication of the HeN11-2B6-P5 mutant was significantly increased in MDCK cells in the late stage of infection (i.e., 48 to 72 h postinfection [hpi]) ( Fig.…”

“…MDCK cells were cultured in DMEM containing 5% FBS. hTERT-PTECs were cultured in DMEM/F-12 medium supplemented with 10% FBS and epithelial cell growth factors (Lonza, Basel, Switzerland), as described previously ( 30 ). A/swine/Henan/11/2005 (H1N1) (HeN11) (GenBank accession numbers HQ541672 to HQ541679 ) was isolated from pigs in China.…”