Development of genetically engineered tet HPV16-E6/E7 transduced human corneal epithelial clones having tight regulation of proliferation and normal differentiation.

Abstract:

: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.

journal_name

Exp Eye Res

authors

Mohan RR,Possin DE,Mohan RR,Sinha S,Wilson SE

doi

10.1016/s0014-4835(03)00175-1

keywords:

subject

Has Abstract

pub_date

2003-10-01 00:00:00

pages

395-407

issue

4

eissn

0014-4835

issn

1096-0007

pii

S0014483503001751

journal_volume

77

pub_type

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