Gene Transfer into Mouse Corneal Epithelial Cell Lines Using an Ecdysone Regulated Ecotropic Retroviral Vector System

Abstract

Abstract: : Purpose: To develop a method for stable gene transfer into a mouse corneal epithelial cell line using ecotropic retroviral vectors. Methods: The cDNA for green fluorescent protein (GFP) subcloned into a retroviral expression vector backbone was co-transfected with expression vectors for the retroviral env and gag-pol structural genes into 293T cells. Viral titer was evaluated by infection of NIH-3T3 cells. Supernatants from these co-transfected cells were used to infect a mouse corneal epithelial cell line, C3H. Expression of GFP was determined by fluorescence microscopy. The cDNA for mouse matrix metalloproteinase-7 (MMP-7) was subcloned into the retroviral vector pCFB-EGSH which contains a promoter regulated by ecdysone-activated steroid binding proteins. The resultant vector pCFB-EGSH-MMP7 and retroviral vector pFB-ERV (encoding the ecdysone binding proteins which activate the pCFB-EGSH promoter in the presence of ecdysone) were co-transfected with env and gag-pol into 293T cells and the virus containing supernatants harvested. MSM-7E cells (immortalized cells from an MMP-7 knockout mouse) were co-infected with the retrovirus containing supernatants in either the presence or absence of 10µM ponasterone A (ponA), an ecdysone analog. Expression of MMP-7 in infected MSM-7E cells with or without ponA induction was evaluated by immunohistochemistry with a polyclonal anti-MMP-7 antibody. Results: The retroviral expression vectors were transfected into 293T cells with an efficiency of approximately 40% as determined by fluorescence microscopy. Supernatants of these transfected cells contained a retroviral titer of approximately 5X10_-6 cfu/ml. Using the retrovirus containing supernatant at a concentration of 5X10_-5 cfu/ml (MOI=1.0) approximately 25% of C3H cells were infected as determined by GFP expression visualized by fluorescence microscopy. MMP-7 was detected by immunohistochemistry in MSM-7E cells infected with pFB-ERV and pCFB-EGSH-MMP7 only when the cells were induced with 10µM ponA. Conclusion: Retroviral vectors provide a novel and efficient method to transfer and express genes in mouse corneal epithelial cell lines. Given the integration of retroviral DNA into the host cell genome this expression should be permanent. We have been able to transfer genes into corneal epithelial cell lines using both constitutive and regulated retroviral gene delivery systems. Further studies will attempt to alter corneal epithelial cell line function using these gene transfer systems.

Keywords: 372 cornea: epithelium • 370 cornea: basic science • 419 gene transfer/gene therapy