Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation.
Lack of an optimal in vitro model of human corneal epithelial (HCE) cells is a major limitation in studying normal functions and gene regulations in HCE. Moreover, availability of a multi-layered HCE culture can reduce the usage of animals in the toxicity testing of consumer products. We have developed tetracycline-responsive human papilloma virus (HPV) 16-E6/E7 transduced HCE clones showing tight regulation of proliferation and normal differentiation. Expression of HPV16-E6/E7 mRNA and HPV16-E7 and keratin K3 proteins was examined by RNase protection assay and western blotting, respectively, in presence and absence (+/-) of Dox in identified clones. Localization of cornea-specific keratin k3 in +/- of Dox was evaluated by immunocytochemistry. The response of growth factors such as hepatocyte growth factor (HGF) and epidermal growth factor to the cellular proliferation in +/- of Dox in the newly identified clones was measured by cell counting. Cellular morphology, formation of multi-layered cultures at air-liquid interface and ultrastructural features were evaluated by light and transmission electron microscopy. The physical barrier established by the newly developed clones was determined by the transepithelial permeability to sodium fluorescein and transepithelial electrical resistance assays in the airlifted-stratified cultures.
Mohan
Rajiv R
RR
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195 6485, USA. rajiv@u.washington.edu
Possin
Daniel E
DE
Mohan
Rahul R
RR
Sinha
Sunilima
S
Wilson
Steven E
SE
D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology; D003315:Cornea / Q000166:cytology* / Q000648:ultrastructure; D019573:Epithelium, Corneal / Q000166:cytology / Q000648:ultrastructure; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D007633:Keratins / Q000235:genetics; D008854:Microscopy, Electron / Q000379:methods; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics
D006133:Growth Substances; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; C059731:oncogene protein E7, Human papillomavirus type 16; D007633:Keratins; D002118:Calcium