Establishment and characterization of an immortalized bovine intestinal epithelial cell line

Primary bovine intestinal epithelial cells (PBIECs) are an important model for studying the molecular and pathogenic mechanisms of diseases affecting the bovine intestine. It is difficult to obtain and grow PBIECs stably, and their short lifespan greatly limits their application. Therefore, the purpose of this study was to create a cell line for exploring the mechanisms of pathogen infection in bovine intestinal epithelial cells in vitro. We isolated and cultured PBIECs and established an immortalized BIEC line by transfecting PBIECs with the pCI-neo-hTERT (human telomerase reverse transcriptase) recombinant plasmid. The immortalized cell line (BIECs-21) retained structure and function similar to that of the PBIECs. The marker proteins characteristic of epithelial cells, cytokeratin 18, occludin, zonula occludens protein 1 (ZO-1), E-cadherin and enterokinase, were all positive in the immortalized cell line, and the cell structure, growth rate, karyotype, serum dependence and contact inhibition were normal. The hTERT gene was successfully transferred into BIECs-21 where it remained stable and was highly expressed. The transport of short-chain fatty acids and glucose uptake by the BIECs-21 was consistent with PBIECs, and we showed that they could be infected with the intestinal parasite, Neospora caninum. The immortalized BIECs-21, which have exceeded 80 passages, were structurally and functionally similar to the primary BIECs and thus provide a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro. In this study, we isolated and cultured primary bovine intestinal epithelial cells and established an immortalized line by transfecting them with a pCI-neo-hTERT recombinant plasmid. Lay Summary In dairy cattle, the intestine is essential for productivity as it contributes nearly 10% of the total metabolizable energy. The intestinal epithelium is at risk of infection from constant exposure to pathogenic microorganisms, which seriously endangers an animal's health, but no bovine intestinal epithelial cell line has been developed so far for research on intestine -related diseases. Thus, the goal of this study was to create an immortalized cell line from isolated primary bovine intestinal epithelial cells. The expression of an exogenous human telomerase reverse transcriptase (hTERT) gene can circumvent the Hayflick limit by maintaining telomere integrity and we used transfection with a plasmid expressing the hTERT gene to convert primary intestinal epithelial cells into an immortalized cell line, which we then characterized. The results showed that the immortalized cell line (BIECs-21) was structurally and functionally similar to the primary bovine intestinal epithelial cells (BIECs) and thus provided a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro.