Development of Targeted Mutations for Loss, Gain, and Partial Attenuation of Rx Homeobox Gene Function

Abstract

Abstract: : Purpose:To develop mouse models for anophthalmia and microphthalmia and to analyze the role of the Rx homeobox gene at various stages of early mammalian retinal development. Methods:Targeting of embryonic stem cells was performed to introduce the RX R192Q mutation, found in a human patient with anophthalmia and sclerocornea, into the mouse Rx gene. A floxed allele of the mouse Rx gene was reported previously. Mice carrying the targeted alleles and the alleles generated via Cre– and FLP–mediated recombination from the original targeted alleles have been used in this study, along with mice carrying the original germline deletion of Rx. Results:In targeting the Rx locus, we have used a standard neomycin–resistance cassette for ES cell selection, flanking it with FLP recognition sequences for easy removal once in the mouse germline. In two distinct targeting reactions, where the neo–cassette was inserted into either intron 1 or 2 of the mouse Rx gene, the neo insert causes null and hypomorphic alleles, respectively. Both alleles are rescued to functional Rx activity levels upon exposure to Flp recombinase, thus serving as conditional activation alleles, adding to the previously generated conditional inactivation allele. Animals homozygous for the hypomorphic allele show a variably penetrant microphthalmia/anophthalmia phenotype, however animals heterozygous for the hypomorphic allele and a null allele are invariably anophthalmic. Upon revomal of the neo insert, animals mimicking the patient phenotype (R192Q/null) show no phenotype, despite having only 5% of the normal Rx DNA–binding activity. Conclusions:We have generated an allelic series of Rx mutations, with the ability to perform conditional activation and inactivation of Rx activity using FLP and Cre recombinases, respectively. Our results suggest that very low levels of Rx activity are sufficient to direct normal eye development, but that at these low levels, a two–fold change in Rx activity has profound effects on eye formation. We are currently using the conditional activation alleles to perform mosaic experiments to determine the cell autonomy of Rx function and using the hypomorphic allele to determine the absolute expression levels required for normal eye development.

Keywords: transgenics/knock-outs • retinal development • genetics