Abstract
Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6tm1Ued (Pax6fl) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6fl/fl and heterozygous Pax6fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6Sey-Neu (Pax6−) null allele. Pax6fl/− compound heterozygotes had more severe eye abnormalities than Pax6+/− heterozygotes, implying that Pax6fl differs from the wild-type Pax6+ allele. Immunohistochemistry showed that the Pax6fl/− corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users.
Highlights
Analysis of genetic mutations has provided key insights in developmental genetics and an allelic series of different types of mutations provides a powerful way of studying gene function
At E14.5 (Fig. 1), the most obvious abnormalities of heterozygous Pax6?/- eyes were that the anterior chamber was not yet formed, as reported previously (Ramaesh et al 2003), and some had an indentation in the middle of the cornea, indicative of a persistent lens stalk
keratin 12 (K12) is normally only expressed in the corneal epithelium and is regulated by Pax6 (Liu et al 1993; Shiraishi et al 1998)
Summary
Analysis of genetic mutations has provided key insights in developmental genetics and an allelic series of different types of mutations provides a powerful way of studying gene function. Many new mouse alleles have been generated by conventional mutagenesis but some have been produced as byproducts when a neomycin-resistance selection cassette (neo cassette) is included in the construction of a floxed allele for Cre-loxP conditional knockout experiments. Sometimes new alleles are deliberately generated in this way but an abnormal phenotype is usually an unwelcome side effect of constructing a floxed allele so the neo selection cassette is often excised. The floxed Pax6tm1Ued allele (abbreviated to Pax6fl) was originally produced to analyse these roles using a conditional knockout approach (Simpson et al 2009) but the possibility that it might produce an abnormal phenotype has not been investigated
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