Preparation of Kera-Cre Transgenic Mice in Which a Floxed Gene is Removed in Keratocytes

Abstract

Abstract: : Purpose: To develop a mouse line in which a gene can be removed in kerotocytes, thus the function of such gene in corneal morphogenesis and homeostasis can be elucidate. Methods: A reporter gene construct containing keratocan promoter and Cre minigene was microinjected into the fertilized mouse eggs to create transgenic mice. The Kera-Cre transgenic mice were crossed with reporter ZEG mice that harbor a transgene containing a chicken ß actin promoter, a floxed lacZ gene (two loxP elements flanked at 5’and 3’ ends of lacZ) and EGFP in tandem. The Cre activity was assessed by the detection of green fluorescence and the loss of ß-galactosidase activity in corneal stroma of compound transgenic mice (Kera-Cre/ZEG). Results: Two independent transgenic mouse lines were obtained. Histochemistry staining with X-gal indicates that in compound Kera-Cre/ZEG mice there was no ß-galactosidase activity and showed green fluorescence. Whereas in simple ZEG transgenic mice the corneal stroma was positive for ß-galactosidase, but negative for green fluorescence. Conclusions: Kera-Cre mice and Cre-loxP system can be of great value in preparing transgenic mouse line to elucidate functions of gene during corneal development and pathophysiology by ablating those genes that have been modified with loxP elements (floxed genes).

Keywords: cornea: stroma and keratocytes • transgenics/knock-outs • gene/expression