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R. de Iongh, S. Lin, H.E. Abud, M.I. Kokkinos, J. Huelsken, M.L. Robinson; Usefulness of Two Cre–Expressing Transgenic Lines for Conditional Deletion of Genes in the Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2790.
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© ARVO (1962-2015); The Authors (2016-present)
Recent transgenic studies have shown that inclusion of a tyrosinase minigene cassette modifies the activity of the αA–crystallin promoter and results in ectopic expression of Cre recombinase in retina and ciliary body (Zhao et al., 2004; IOVS 45, 1930–9). To investigate the potential usefulness of these mice for studies of retinal development, we investigated the spatiotemporal activity of Cre in the retina of two lines of transgenic mice and the efficacy of the retinal Cre to delete a floxed gene, ß–catenin (CatnB).
Cre–expressing mice (MLR34 and MLR37) were crossed to ‘floxed’ ß–catenin mice, which also carry a Cre reporter (LacZ) transgene. Ocular tissues from F1 progeny at various stages of development (E13–P21) were stained for ß–galactosidase (LacZ) to detect Cre activity. Mutant mice (Cre+/CatnBLoxP/LoxP), generated by F1 intercrosses were identified by PCR. Postnatal retinae from mutant and wild–type mice were analysed by PCR, histology and immunofluorescence.
In both lines of mice, Cre was first detected at E13.5, in groups of neuroblasts along the inner margin of the peripheral optic cup. By E15.5, expression included parts of the outer neuroblastic layer. At P2–4, Cre was detected in columns of cells in the retina, extending from the nerve fiber to the neuroblastic layer. At P21, Cre activity increased but was restricted to the inner retina (nerve fibre to outer plexiform layer) with little or no activity in the outer nuclear layer. Cre activity patterns were variable among retinae. Neither line showed Cre activity in the ciliary margin during development. Comparison of wild–type and mutant retinae revealed no gross morphological abnormalities. PCR of retinal DNA revealed successful recombination and deletion of at least one allele in some cells. However, double–labelling for ß–catenin and ß–galactosidase revealed that, despite intense staining for Cre activity, there was little or no detectable loss of ß–catenin protein in mutant retinae.
The clonal expression of Cre within the retinae of these mice raises the possibility of studying cells with Cre–mediated gene mutations adjacent to wild type cells in the same retina. However, levels of Cre appear to be insufficient for deleting two floxed alleles. Future studies will determine the efficacy of deleting only one floxed allele in mice heterozygous for floxed and null ß–catenin alleles.
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