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Natalie Dora, J. Martin Collinson, Robert E Hill, John D West; Genetic background effect on eye phenotype of Le-Cre transgenic mice. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6400.
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Le-Cre transgenic mice (Ashery-Padan et al 2000, Genes Dev 14:2701-11) express Cre-recombinase in the developing head surface ectoderm from Pax6-Le tissue-specific regulatory elements. They have been widely used in Cre-loxP experiments to delete floxed genes in the developing surface ectoderm. We planned to use Le-Cre mice to deplete Pax6 only in surface ectoderm derivatives and compare the corneal epithelial phenotype to our previous studies with Pax6+/- mice, where Pax6 is depleted globally. Our first hypothesis was that crossing the Le-Cre transgene to the CBA/Ca inbred strain would not compromise its action and use of the same genetic background would make direct comparisons with our previous studies of CBA/Ca-Pax6+/- mice more meaningful.
Hemizygous Le-Cre and heterozygous floxed Pax6fl/+ mice were each crossed to inbred CBA/Ca mice for several generations and then intercrossed to produce experimental Le-Cre;Pax6fl/+ mice and three control genotypes. A panel of phenotypic endpoints was investigated to determine the effects of tissue specific depletion of Pax6 on the eye phenotype.
Preliminary results were promising and adult Le-Cre;Pax6fl/+ corneas were abnormal. Eyes were small, the corneal epithelium had fewer cell layers, goblet cells accumulated and Pax6 & K12 immunostaining levels were reduced. As expected, WT;Pax6fl/+, Le-Cre;WT and WT;WT controls were normal. We analysed controls at each generation and, after several CBA/Ca backcross generations, Le-Cre;WT controls also had abnormal eyes, with phenotypes similar to those caused by Pax6-depletion. We started to breed out the CBA/Ca background by crossing Le-Cre mice to the original FVB strain and the Le-Cre;WT phenotype began to normalise.
Eye abnormalities occur in some homozygous Le-Cre mice and genetic background affects penetrance (Robinson et al 2005, IOVS 46, E-abstr. 1885). Our study now shows that genetic background-dependent eye abnormalities can also occur in hemizygous Le-Cre mice. This may have significant implications for other studies. Offspring with deleted floxed alleles are analysed in most Le-Cre experiments but these are also hemizygous for Le-Cre and often on mixed genetic backgrounds. Furthermore, many published Cre-loxP studies give incomplete details about which controls are analysed. Our results highlight the importance of including all the relevant controls in Cre-loxP experiments.
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