human VMD2 promoter to direct the expression of a reverse tetracycline-inducible system controlled Cre recombinase in transgenic mice. However, functional analysis of these transgenic mice demonstrated that Cre activity was not exclusively present in the RPE. In addition to the RPE, Cre function was also identified in other retinal cells, including Müller cells. In this report we summarize the expression pattern of two VMD2-cre mouse lines that demonstrated Cre activity specifically in the RPE or predominantly in the Müller cells.

24.2 Materials and Methods

24.2.1 Experiment with Animals

Transgenic mice were generated as described previously (Le et al. 2008). All animal procedures were performed according to the guidelines of the ARVO statement for the ‘Use of Animals in Ophthalmic and Vision Research’ and were approved by the Institutional Animal Care and Use Committees at the University of Oklahoma Health Sciences Center, the Dean A. McGee Eye Institute, and the Oklahoma Medical Research Foundation. PCR analysis diagnostic for cre, rtTA and Creactivatable lacZ genes was carried out, as described previously (Le et al. 2008). Doxycycline was administered through the drinking water of pregnant mothers at a concentration of 0.5 mg/mL in 5% sucrose.

24.2.2 β-Galactosidase Assay

β-Galactosidase assay was carried out using cryprotected retinal sections. Briefly, eyes were fixed in 2% paraformaldehyde and 0.25% glutaraldehyde in phosphatebuffered saline (PBS) at 4◦C for 1 h. The lens was then removed and the posterior part of the eye was cryoprotected in 30% sucrose, frozen in OCT medium, and sectioned. Retinal sections (10 μm) were briefly fixed in 0.5% glutaraldehyde for 5 min, washed with PBS 3 times, and incubated in X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactoside) staining solution (1 mg/ml X-gal, 6 mM potassium ferricyanide, 6 mM potassium ferrocyanide, 2 mM MgCl2, 0.02% NP-40, and 0.01% sodium deoxycholate in 1X PBS) at room temperature for 3–12 h, depending on experimental requirements. Sections were then cover-slipped and observed under a microscope.

24.3 Results

24.3.1 Generation of Transgenic Mice

Human VMD2 gene is thought to be expressed preferentially in the RPE (Marquardt et al. 1998; Petrukhin et al. 1998) and has been used to direct transgene expression in the RPE (Esumi et al. 2004). To generate tetracycline-inducible RPE-specific Cre