Abstract
Abstract: :
Purpose: Our laboratory has demonstrated that matrix metalloproteinase–7 (MMP–7) plays an antiangiogenic role in the cornea. Mice deficient for MMP–7 have increased neovascularization following excimer laser wounding. We are developing a retroviral vector system to express MMP–7 in cell lines derived from MMP–7 knockout mice. Methods: Immortalized cell lines derived from MMP–7 deficient mice were genotyped using PCR and immunostained for MMP–7 to confirm both the absence of the MMP–7 gene and any detectable MMP–7 protein. The cDNA for proMMP–7 as well as the cDNA for GFP were subcloned into a MMLV–derived retroviral bi–cistronic expression vector. Infective retroviral particles were obtained by co–transfection of HEK–293T cells with the above construct in conjunction with gag–pol and env (ecotropic) expressing plasmids. Retroviral containing supernates were used to infect cornea–derived keratocytes and epithelium. Pure populations of infected cells were enriched using fluorescence activated cell sorting (FACS). Infected cell were characterized for heterologous gene expression using a combination of fluorescent microscopy and immunostaining Results: Immortalized cell lines from MMP–7 deficient mice were genotypic as well as functional MMP–7 knockouts as determined by genomic PCR and immunohistochemistry. HEK–293T cells were efficiently co–transfected with expression plasmids (70%) as determined by GFP expression. Cornea–derived cells infected with the bi–cistronic proMMP–7/GFP vector expressed the transgene (approximately 20%) as detected by GFP expression. Following FACS, 100% of cells expressed the construct. Immunohistochemistry for MMP–7 confirmed the expression of MMP–7 by the infected cell lines. Conclusions: The MMP–7 gene, an antiangiogenic protein expressed in the cornea, can be efficiently transferred to cornea–derived cell lines. Using a bi–cistronic expression vector, co–expressing proMMP–7 and GFP, facilitates both the monitoring of gene expression as well as the enrichment of infected cells by FACS.
Keywords: cornea: stroma and keratocytes • cornea: basic science • gene transfer/gene therapy