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VA Voronina, SV Kozlov, PH Mathers, M Lewandoski; Conditional Inactivation Of The Rx Homeobox Gene Results In Viable Anophthalmic Animals . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2003.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Conventional gene targeting of the murine Rx gene results in birth of anophthalmic pups that die within the first day due to a variety of neural defects. Based on this finding and Rx expression in the developing and adult retina, we set out to elucidate the function of the Rx gene at later stages through conditional gene inactivation. Methods: We generated a Rx targeting vector with exon 2 flanked by two loxP sites (floxed) and neomycin resistance gene flanked by frt sites. This vector was electroporated into embryonic stem cells and correctly targeted clones were injected into mouse blastocysts. Chimeric animals were bred to produce allelic series at the Rx locus. One allele was used for the conditional inactivation of Rx in the retina. Results:We generated mice that contain an allelic series at the Rx locus: a null allele, a hypomorphic allele (due to insertion of neomycin resistance gene in the intron 1) and a conditional allele with wild-type activity but subject to Cre-mediated inactivation. The null homozygotes recapitulate the previously published phenotype, including early postnatal lethality, anophthalmia, brain defects and cleft palate. Hypomorphic homozygotes and compound heterozygous (carrying a null and a hypomorphic allele) are anophthalmic and die soon after birth. However, these animals are less severely affected because we did not observe any obvious brain defects. Removal of the neomycin resistance gene from the hypomorphic allele results in conditional allele. Animals homozygous for this allele or heterozygous for this allele and the null allele are indistinguishable from wild type littermates. To specifically knockout Rx in the retina, the conditional allele was inactivated in mice carrying the Foxg1-Cre transgene, which is expressed in the anterior neural ridge at E8.5 and later in the nasal retina. These animals lack eyes and optic nerves but otherwise appear to be normal. Conclusion: Conditional Rx inactivation in the developing retina results in birth of viable anophthalmic animals. This phenotype makes them an ideal model for true anophthalmia as seen in the patient population.
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