Generation of Cre Transgenic Mice with Postnatal RPE-Specific Ocular Expression

The cre-loxP system has revolutionized mouse genetics by facilitating the creation of cell-type–specific knockout animal models. Cre recombinase is a P1 bacteriophage protein that binds to a 34-bp-long target recognition site known as loxP.1 Through intramolecular recombination, cre has the ability to excise loxP-flanked (floxed) sequences from the genome, leaving a single loxP site in its place. Tissue-specific promoter-driven cre expression makes possible the study of cell-autonomous gene function and permits the examination of genes whose systemic absence is lethal. Such a system is of particular interest in the study of diseases of the eye due to the complex interplay of the various cell types that make up the retina. The retinal pigment epithelium (RPE) is a monolayer lying between the neural retina and Bruch's membrane. It performs many specialized functions that support and nourish the photoreceptors, such as maintaining the blood–retina barrier by regulating the movement of nutrients, ions, and water between the photoreceptors and the choroid; the phagocytosis of shed photoreceptor outer segments; and the facilitation of retinoid metabolism (visual cycle).2 In addition, human RPE dysfunction has been implicated in the pathogenesis of both wet and dry age-related macular degeneration, the most common cause of irreversible blindness in the elderly populations of developed countries.3–5 Four mouse lines that express cre in the RPE have been reported.6–9 Two of the lines that express it in the RPE—a dopachrome tautomerase (Dct)-cre line7 and a tyrosinase related protein line (TRP)-1-cre6—do so in a noninducible manner and exhibit cre expression and activity during embryonic development. These lines were not assessed for effects of cre expression on retinal morphology or function in adult animals. A third line utilizes an inducible monocarboxylate transporter 3 promoter to drive RPE-specific cre expression.8 When crossed with a Rosa-lacZ line, cre expression occurs in approximately 20% of RPE cells. When crossed with a cre-activated diphtheria toxin line, the number of missing RPE cells suggests a higher percentage of cre-expressing RPE cells, but this has not been directly demonstrated. Retinal structure was shown to be normal 6 months after induction. Recently, a mouse line that takes advantage of the tetracycline-inducible system to control cre expression has been developed for the knockout of genes from the RPE.9 The reverse Tet-inducible transactivator is under control of a 2.9-kb fragment of the bestrophin 1 (BEST1) promoter, whereas cre is located downstream of the (tetracycline-responsive element [TRE]), which, in theory, should limit cre expression to the RPE and only when the animal has been given doxycycline. Maximal cre activity was achieved after induction at P4, but substantial activity was detected on induction as late as P25. No cre-mediated retinal toxicity was observed up to 10 months of age. This inducible BEST1-cre mouse line is likely to be a useful tool; however, the complication of daily doses of doxycycline, which is done by gavage in animals before weaning, may limit the utility of this line for some applications. We therefore sought to generate a transgenic mouse line with constitutive RPE-restricted expression of cre beginning after ocular development for use in studying RPE function in the developed eye and age-related retinal disease. We chose to use a fragment of the human BEST1 promoter which has been shown to promote robust ocular expression that is restricted to the RPE in the eye of transgenic mice.10 Herein, we provide analysis of a new BEST1-cre transgenic line in which we study cre expression timing, localization, enzymatic activity, and effect on retinal integrity during the full mouse lifespan.