Isolation and characterization of an immortalized oral keratinocyte cell line of mouse origin

Abstract

Objective-To establish an oral epithelial cell line of mouse origin for molecular and biochemical assays.Design-Epithelial cells were isolated from the oral cavity of adult mice and established as a spontaneously immortalized cell line in culture, designated immortalized oral keratinocyte cells (IMOK cells). The cells were then characterized for growth characteristics, differentiation potential, karyotype, transfectability, susceptibility to viral infection and responses to siRNA.Results-The IMOK cells exhibit robust growth in both serum-containing and serum-free medium for at least 100 population doublings. IMOK cells have a near diploid karyotype, express keratinocyte marker proteins and can be induced to undergo differentiation by addition of high levels of calcium to the medium. The differentiation process is characterized by morphological changes and by the induction of oral epithelium specific differentiation marker sproteins such as K4 and K13. Transient transfection analyses reveal that IMOK cells are highly transfectable and that several promoters of epithelial cells are active in these cells. Moreover, upon differentiation with calcium, there is an upregulation of differentiation-specific K4 and Elf5 promoter activity. Finally, we show that the oral keratinocytes are also amenable to infection with retroviruses and to siRNA based knock-down of gene expression.Conclusions-Our study is the first to establish an immortalized oral keratinocyte cell line of murine origin that can recapitulate the oral epithelium differentiation program and thus could serve as a useful tool for toxicological and molecular analyses of the oral tissue.

Glucocorticoid Receptor Regulates Overlapping and Differential Gene Subsets in Developing and Adult Skin

“…These proteins are normally detected in the cornified envelope (CDSN) and upper layers of the developing skin (PTGS1, BCL6) and have been previously reported to be involved in keratinocyte differentiation (29,30,25). ELF5 expression in skin is controversial because some reports describe Elf5 expression in mouse epidermis and oral keratinocyte cell lines (26,31) whereas others describe Elf5 exclusively in the inner root sheath of the hair follicle (32). To better understand the contribution of the identified genes in epidermal development, we focused on a subset of the novel GR targets, using the previously described Dex-regulated genes as a control of transcriptional regulation mediated by GR in vivo.…”

“…In A549 cells, the majority of GBS (63%) were found to be more than 10 kb distal from transcriptional start sites (31). This explains the difficulty of identifying GR transcriptional targets by conventional approaches searching for consensus GBS sequences in proximal promoters.…”

“…It was recently shown that most GBS in the natural gene context are represented by nonconventional GRE sites and/or composite elements that are distributed evenly through the genomic DNA (3,31,32). In A549 cells, the majority of GBS (63%) were found to be more than 10 kb distal from transcriptional start sites (31).…”

Establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line

“…The chromosomes of Passage-50 hTERT-CBECs were examined following standard methods described by Parikh et al 2008, with slight modifications. Briefly, the cells were initially cultured in T25 cell culture flasks (Thermo Fisher, Waltham, USA) containing D/F12 supplemented with 2 % FBS plus the growth factors described above for 24-36 h, treated with 0.05 μg/mL colcemid (Aladdin, Shanghai, China) for 3 h at 37°C, trypsinized, spun down, and then incubated in 0.075 mol/L KCI for 20 min at 37°C.…”

“…The airway epithelium has been shown not only to serve as a physicochemical barrier against inhaled chemical or physical substances and infectious agents but also to be involved in the pathogenesis of respiratory diseases in humans and animals (Abraham et al 2011). Many studies have described host-pathogen interactions in respiratory epithelial cell cultures derived from humans (Vos et al 2005) and from a variety of animals (Wang et al 2014), including mice (Parikh et al 2008), horses (Abraham et al 2011), cattle (Su et al 2013), and others. Airway epithelial cells have been widely used as a cell model in studies of the pathogenesis of human, murine, or swine respiratory pathogen infections (Massin et al 2010).…”