Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profile of the SV40-immortalized human corneal epithelial cells

ABSTRACT: Microarray was used to study global gene expression of a cell culture model based on SV40-immortalized human corneal epithelial (iHCE) cells. The gene expression profile of the cell line was compared to the normal human corneal epithelium. Affymetrix HG-U133A GeneChipsÂ® were used for microarray experiments and results were validated by performing RT-qPCR for selected genes. iHCE was found to over- and under-express 22 % and 14 % of the annotated genes, respectively. The results of this study suggest that differences between iHCE cells and normal corneal epithelium are substantial and therefore the use of these cells in corneal research should be considered with caution. SV40-immortalized human corneal epithelial (iHCE) cells were cultured on collagen coated permeable supports for one week using standard culture conditions. Subsequently the apical medium was removed and culturing was continued at the air-liquid interface which allows cells to stratify. Trans-epithelial electrical resistance was followed and total RNA was extracted from cultures with resistance > 300 Î© x cm2. RNA was amplified, labeled with biotin, fragmented and hybridized to the HG-U133A GeneChipsÂ® (Affymetrix) according to the protocol of the manufacturer. Three separate experiments with samples from three independent culture batches (iHCE1, iHCE2, iHCE3) were performed. The Global gene expression profile of iHCE was compared to previously published human corneal epithelial tissue (GSE5543) data.

ORGANISM(S): Homo sapiens

SUBMITTER: Petri Auvinen

PROVIDER: E-GEOD-22539 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression analysis in SV-40 immortalized human corneal epithelial cells cultured with an air-liquid interface.

Greco Dario D Vellonen Kati-Sisko KS Turner Helen C HC Häkli Marika M Tervo Timo T Auvinen Petri P Wolosin J Mario JM Urtti Arto A

Molecular vision 20101015

<h4>Purpose</h4>To compare the global gene expression profile of stratified epithelia generated in vitro using simian virus 40 (SV40) immortalized human corneal epithelial cells with the previously reported gene expression of normal human corneal epithelia.<h4>Methods</h4>Immortalized cells expanded in submerged culture were grown in an air-liquid interface of liquid permeable collagen-coated filters to foster stratification and differentiation. Stratified epithelia displaying resistances exceed ...[more]

PMID: 21139686

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Project description:Purpose. Simian virus 40 (SV40)-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. To better understand the nature of this cell line, we assessed it for genomic aberrations and cellular heterogeneity. Methods. For the quantitative measurement of genomic aberrations, array based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, cell morphology, growth kinetics, trans-epithelial electrical resistance and transcriptional efficiency were analyzed. We also carried out real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci in order to assess genomic heterogeneity. To assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells, we collected expressed sequence tags (ESTs) for this cell line. Southern blotting and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG). Results. Array CGH analysis indicated that the genomic content of HCE-T cells is different from the normal healthy genome. Our results from cellular functional assays, real-time PCR and cFISH strongly showed that HCE-T cells consist of a not insignificant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9. Conclusions. Our results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies which use this cell line. Keywords: comparative genomic hybridization Genomic DNAs from HCE-T cells, immortalized cell line of human corneal epithelial cells and normal female duploid cells were subjected to array CGH analysis to identify genomic alterations in the HCE-T cells. These DNAs were labeled with Cy5 and Cy3, respectively.

Dataset Information

Gene expression profile of the SV40-immortalized human corneal epithelial cells

Publications

Gene expression analysis in SV-40 immortalized human corneal epithelial cells cultured with an air-liquid interface.

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