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      Analysis of compound heterozygotes reveals that the mouse floxed Pax6 tm1Ued allele produces abnormal eye phenotypes

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          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 Pax6 tm1Ued ( Pax6 fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 fl/fl and heterozygous Pax6 fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 Sey- Neu ( Pax6 ) null allele. Pax6 fl/ compound heterozygotes had more severe eye abnormalities than Pax6 + / heterozygotes, implying that Pax6 fl differs from the wild-type Pax6 + allele. Immunohistochemistry showed that the Pax6 fl/ corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 fl 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.

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          The online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users.

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          Most cited references63

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          Mouse small eye results from mutations in a paired-like homeobox-containing gene.

          Small eye (Sey) in mouse is a semidominant mutation which in the homozygous condition results in the complete lack of eyes and nasal primordia. On the basis of comparative mapping studies and on phenotypic similarities, Sey has been suggested to be homologous to congenital aniridia (lack of iris) in human. A candidate gene for the aniridia (AN) locus at 11p13 has been isolated by positional cloning and its sequence and that of the mouse homologue has been established (C.T., manuscript in preparation). This gene belongs to the paired-like class of developmental genes first described in Drosophila which contain two highly conserved motifs, the paired box and the homeobox. In vertebrates, genes which encode the single paired domain as well as those which express both motifs have been described as the Pax multigene family. A Pax gene recently described as Pax-6 is identical to the mouse homologue of the candidate aniridia gene. Here we report the analysis of three independent Sey alleles and show that indeed this gene is mutated and that the mutations would predictably interrupt gene function.
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            An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination.

            We describe a strategy for generating an allelic series of mutations at a given locus that requires the production of only one targetted mouse line. The 'allelogenic' mouse line we produced carries a hypomorphic allele of Fgf8, which can be converted to a null allele by mating to cre transgenic animals. The hypomorphic allele can also be reverted to wild-type by mating the allelogenic mice to flp transgenic animals, thereby generating a mouse line suitable for Cre-induced tissue-specific knockout experiments. Analysis of embryos carrying different combinations of these alleles revealed requirements for Fgf8 gene function during gastrulation, as well as cardiac, craniofacial, forebrain, midbrain and cerebellar development.
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              PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects.

              The human eye malformation aniridia results from haploinsufficiency of PAX6, a paired box DNA-binding protein. To study this dosage effect, we characterized two PAX6 mutations in a family segregating aniridia and a milder syndrome consisting of congenital cataracts and late onset corneal dystrophy. The nonsense mutations, at codons 103 and 353, truncate PAX6 within the N-terminal paired and C-terminal PST domains, respectively. The wild-type PST domain activates transcription autonomously and the mutant form has partial activity. A compound heterozygote had severe craniofacial and central nervous system defects and no eyes. The pattern of malformations is similar to that in homozygous Sey mice and suggests a critical role for PAX6 in controlling the migration and differentiation of specific neuronal progenitor cells in the brain.
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                Author and article information

                Contributors
                John.West@ed.ac.uk
                Journal
                Transgenic Res
                Transgenic Res
                Transgenic Research
                Springer International Publishing (Cham )
                0962-8819
                1573-9368
                30 May 2016
                30 May 2016
                2016
                : 25
                : 5
                : 679-692
                Affiliations
                [1 ]Genes and Development Group, Centre for Integrative Physiology, Biomedical Sciences, University of Edinburgh Medical School, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK
                [2 ]Genes and Development Group, Centre for Integrative Physiology, Clinical Sciences, University of Edinburgh Medical School, Hugh Robson Building, George Square, Edinburgh, EH8 9XD UK
                [3 ]Institute for Adaptive and Neural Computation, School of Informatics, University of Edinburgh, 10 Crichton Street, Edinburgh, EH8 9AB UK
                [4 ]Biomathematics and Statistics Scotland, James Clerk Maxwell Building, Peter Guthrie Tait Road, Edinburgh, EH9 3FD UK
                Article
                9962
                10.1007/s11248-016-9962-4
                5023747
                27240603
                fca2e6fc-3bf4-4f04-bab4-fbe83735f38f
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 22 December 2015
                : 13 May 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 088876/Z/09/Z
                Award Recipient :
                Funded by: Medical Research Council (UK)
                Award ID: J003662
                Award ID: G0800429
                Award Recipient :
                Categories
                Original Paper
                Custom metadata
                © Springer International Publishing Switzerland 2016

                Genetics
                pax6,floxed allele,eye,corneal epithelium,keratin 12
                Genetics
                pax6, floxed allele, eye, corneal epithelium, keratin 12

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