Topical Gene Ablation of sflt-1 Abolishes Corneal Avascularity

Abstract

Purpose:: To test the hypothesis that corneal avascularity is maintained by soluble VEGFR-1 (sflt-1) by means of a novel method of conditional gene ablation effected by an eye drop preparation of Cre recombinase linked to a nuclear localization sequence (NLS-Cre).

Methods:: flt1 loxP/loxP mice on a C57Bl/6 background and wild-type controls as well as ROSA26R lacZ reporter mice were studied. Corneal expression of flt-1, sflt-1, and Cre were examined by immunohistochemistry and western blotting. NLS-Cre or NLS-ß-gal was applied topically with or without prior application of proparacaine. Cornea flat mounts were stained with CD31-FITC to detect blood vessels.

Results:: The avascular cornea expressed sflt-1 while the vascular conjunctiva which expressed the cell surface flt-1. Topical NLS-Cre delivered Cre into the nuclei of corneal epithelial cells within 1 hour of application and was augmented by proparacaine in wild-type mice, effected recombination in lacZ reporter mice, and abolished corneal avascularity in flt1 loxP/loxP but not wild-type mice. flt1 loxP/loxP and wild-type mouse corneas treated with NLS-ß-gal remained avascular.

Conclusions:: In vivo suppression of sflt-1 by conditional gene ablation terminates corneal avascularity demonstrating a non-redundant role for sflt-1 in this process. Topical tissue-specific gene ablation is a significant addition to the arsenal of gene manipulation. This novel approach elides the drawbacks of the traditional Cre-lox system (toxicity of constitutive Cre expression, leaky or weak promoters, time consuming interbreeding) while conferring exquisite spatial and temporal selectivity: a strategy sure to find broad applicability in the future.

Keywords: cornea: basic science • gene/expression • receptors