Toward Temporal and Spatial Control of Retinal Gene Expression

Abstract

Abstract: : Purpose: The Pdeg_tm1/Pdeg_tm1 mutant mouse (carrying a targeted deletion of the gene encoding PDEγ, Pdeg) lacks PDE activity and undergoes rapid retinal degeneration. An interaction between the inhibitory PDEγ and catalytic PDEαß subunits may be critical for the proper folding, conformation or stabilization of the PDE enzyme complex. Unfortunately, the maldeveloped and truncated outer segments of the Pdeg_tm1/Pdeg_tm1 mouse prohibit their use in biochemical studies. A fully developed photoreceptor that lacks PDEγ will allow us to test if PDEγ has a positive role in the formation of an active PDE complex. We will develop an inducible gene expression system that will make possible to delete or activate genes in an adult mouse. Methods: Fluorescence microscopy was used to monitor floxed-Pdeg--IRES-EGFP expression in the retina of mice carrying a modified allele of the PDEγ gene (Pdeg). EGFP expression in single rods was detected in the suction electrode after stimulation at 488 nm. Results:Transgenic mouse lines were established using embryonic stem cells carrying a Pdeg floxed allele with an IRES-EGFP cassette introduced into its 3' end. To test the expression of this construct in 293T cells, in vitro, a CMV promoter was placed at its 5' end. EGFP fluorescence was detected after transfecton. The floxed-Pdeg-IRES-EGFP cassette (without the CMV promoter) was then introduced into the mouse germline. Sections of retinas from the resulting mice showed that EGFP was specifically expressed in photoreceptor cells. In vivo, Cre-mediated site-specific recombination should result in loss of both Pdeg and EGFP expression. Conclusion:We have successfully used EGFP as a reporter for the expression of PDEγ. The lines of floxed mice that we have generated will be utilized to monitor Cre-mediated recombination in photoreceptors. An inducible gene targeting system may allow us to address several previously unapproachable problems in sensory biology.

Keywords: 562 retinal degenerations: hereditary • 316 animal model • 517 photoreceptors