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Arlene A. Hirano, Jim Boulter, Nicholas C. Brecha; Selective Gene Targeting Of Horizontal Cells: Generation Of A Connexin-57-iCre Transgenic Mouse. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4113.
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Horizontal cells mediate inhibitory feedback in the outer retina; however, the cellular mechanisms that underlie this neurotransmission are poorly understood. To fill this gap, we generated transgenic mice in which cre recombinase (cre) gene expression is driven by connexin57 (Cx57) regulatory elements, such that cre is expressed in horizontal cells.
In the targeting construct, the protein-coding region of the Cx57 gene was replaced by the ‘improved’ cre gene (iCre) and contained a neomycin (neo) resistance cassette flanked by two Flp recombinase recognition (frt) sites upstream of the iCre gene. The construct was electroporated into ES cells, and the resultant neo-resistant clones screened for homologous recombination. The successfully targeted ES cell clones were injected into mouse blastocysts (E3.5) at the UCLA ES Cell and Transgenic Animal Facility. The resultant chimeric male pups were crossed with C57BL/6 wildtype female mice, and the progeny scored for germline transmission. Retinas were examined by indirect immunofluorescence and confocal microscopy.
Four out of 12 chimeric males showed germline transmission. Double labeling of vertical sections of heterozygous and homozygous Cx57-iCre and wild-type retinas with antibodies to calbindin and cre showed that the cre protein was restricted to the nuclei of horizontal cells in the outer retina. Crossing of Cx57-iCre mice with a ROSA26-tdTomato cre reporter mouse resulted in tdTomato fluorescence in retinal horizontal cells, indicating that iCre is functional and can mediate recombination. Double labeling of whole-mount retinas indicated that cre is present in horizontal cells in all areas of the retina. This finding was confirmed by examination of whole-mount retinas of Cx57-iCre x ROSA26-tdTomato mice.
By genetically engineering a transgenic mouse that expresses cre selectively in horizontal cells, we have developed an experimental tool that will enable us to manipulate proteins in these cells by crossing the Cx57-iCre mouse with the growing number of mouse lines containing floxed genes, to study the role of horizontal cells in visual processing.
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