title: Establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line.
abstract: Dogs are susceptible to infectious diseases that occur primarily in the respiratory tract. The airway epithelium acts as a first line of defense and is constantly exposed to microorganisms present in the environment. Respiratory epithelial cells have recently gained wide use as a cell model for studying the pathogenesis of human, murine or swine respiratory pathogen infections. However, studies of the pathogenic mechanisms of canine pathogens have been hindered by the lack of reliable respiratory cell lines. Here, we cultured primary canine bronchiolar epithelial cells (CBECs), whose characteristics were confirmed by their expression of the epithelial cell-specific marker cytokeratin 18, and have provided protocols for their isolation and ex vivo expansion. Further, we established immortalized CBECs containing the human telomerase reverse transcriptase (hTERT) gene via transfection of primary CBECs with the recombinant plasmid pEGFP-hTERT. Immortalized bronchiolar epithelial cells (hTERT-CBECs) retain the morphological and functional features of primary CBECs, as indicated by reverse transcriptase polymerase chain reaction, proliferation assays, karyotype analysis, telomerase activity assay, and Western blotting, which demonstrate that hTERT-CBECs have higher telomerase activity, an extended proliferative lifespan, and a diploid complement of chromosomes, even after Passage 50. Moreover, this cell line is not transformed, as evaluated using soft agar assays and tumorigenicity analysis in nude mice, and can therefore be safely used in future studies. The isolation and establishment of stable hTERT-CBECs is of great importance for use as an in vitro model for mechanistic studies of canine pathogenic infections.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Xie
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Maoda
authors.initials: M
authors.lastname: Pang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Liang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Yanbing
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Ma
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Dildar Hussain
authors.initials: DH
authors.lastname: Kalhoro
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Chengping
authors.initials: C
authors.lastname: Lu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. liuyongjie@njau.edu.cn.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D004171:Diploidy; D004285:Dogs; D004847:Epithelial Cells / Q000201:enzymology / Q000502:physiology*; D015870:Gene Expression; D042822:Genomic Instability; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D019098:Telomerase
keywords: Canine bronchiolar epithelial cells (CBECs); Cell model; Pathogenesis; hTERT-CBECs