Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

Kengo Kuroda,Emiko Isogai,Katsuya Okuno,Tohru Kiyono,Mizuki Masuda,Tomokazu Fukuda,Yukako Koyanagi,Moe Narita,Gabriele Saretzki

doi:10.1371/journal.pone.0143473

Abstract

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.

Highlights

Cattle are the economically important domestic animal source of livestock-related products such as meat and milk
We named the bovine fetal colon epithelial (BFCE) cells transduced with mutant cyclin dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) as “BFCE-K4DT” (Bovine Fetal Colon Epithelial cell established with CDK4, cyclin D1 and TERT)
As a solution to this limitation, we introduced human Cyclin D1, mutant CDK4, and TERT

Summary

Introduction

Cattle are the economically important domestic animal source of livestock-related products such as meat and milk. The efficiency of their production is largely affected by various zoonotic. Establishment of Bovine Colon Epithelial Cell Line pathogens such as Escherichia coli O157:H7 [1] and Salmonella enterica [2]. Many pathogens in cattle are living as commensal bacteria at the mucosal surface without invading the reservoir host, exponential growth of the bacteria and invasion into the intestinal epithelial cells are critical steps to establish infection in infected animals. An established bovine colon epithelial cell line would be a powerful tool for studies that assess the effects of infectious bacteria on host colon epithelial cells

Methods

Results

Conclusion