Establishment of a bovine rumen epithelial cell line.

Abstract

Rumen epithelium plays an essential role in absorption, transport, and metabolism of short-chain fatty acids, the main products of rumen fermentation, and in preventing microbes and other potentially harmful rumen contents from entering the systemic circulation. The objective of this study was to generate an immortal rumen epithelial cell line that can be used as a convenient model of rumen epithelial cells in vitro. We isolated primary rumen epithelial cells from a steer through trypsin digestion and transduced them with lentiviruses expressing the Simian Virus (SV) 40 T antigen. We cloned the transduced cells by limiting dilution. Western blotting analysis confirmed the expression of the SV40 T antigen in two single-cell clones. Cells from one clone, named bovine rumen epithelial clone 1 (BREC1), displayed a flat and squamous morphology in culture. RNA sequencing revealed that BREC1 cells expressed many markers of epithelial cells, including keratins, the epidermal growth factor receptor, and the short-chain fatty acid transporters monocarboxylic acid transporter (MCT) 1 (MCT-1) and MCT-4. RNA sequencing revealed that BREC1 cells expressed key enzymes such as 3-hydroxymethyl-3-methylglutaryl-CoA lyase and 3-hydroxy-3-methylglutaryl-CoA synthase 1 involved in ketogenesis, a unique function of rumen epithelial cells. RNA sequencing also revealed the expression of genes encoding tight junctions, desmosomes, anchoring junctions, and polarized plasma membranes, structures typical of epithelial cells, in BREC1 cells. Cell proliferation assays indicated that BREC1 cells were similar to primary rumen epithelial cells in response to insulin-like growth factor 1, insulin, and butyrate. In conclusion, BREC1 is not only a convenient but an appropriate model for studying the factors and mechanisms that control proliferation, apoptosis, differentiation, nutrient transport, metabolism, and barrier function in rumen epithelium.