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B. Bakall, P. J. McLaughlin, J. B. Stanton, H. C. Hartzell, L. Y. Marmorstein, A. D. Marmorstein; The Bestrophin-2 Knock-Out Mouse: Histological and Functional Analysis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2983.
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To determine in which tissues and cell types best-2 is expressed and to examine the functional implications of a lack of best-2 expression in the mouse.
The Vmd2L1 gene was disrupted in mice by replacing the first two exons of the gene with a LacZ construct and confirmed by Southern blot and RT-PCR. Best-2 mRNA expression was assessed by staining of fixed sections of various organs with X-gal. Tissues which were positive for gene expression were examined by immunohistochemistry using a polyclonal mouse anti-best-2 antibody. As best-2 is reportedly expressed in olfactory epithelia, olfactory function was tested using a behavioral assay. The reported research was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
The mouse Vmd2L1 gene was successfully disrupted resulting in knock-out mice, verified by Southern Blot and RT-PCR of whole eyes. No mouse best-2 was detected by immunohistochemistry in homozygous knock-out mouse eyes. Mice were viable with no gross anatomic or histologic abnormalities noted. Consistent with prior studies using RT-PCR, X-gal staining of multiple organs identified eye and colon as primary sites of best-2 expression. X-gal positive cells were identified in nasal tissues after prolonged incubation (overnight) with substrate, however, similar non-specific staining was observed in wt controls. Best-2 protein expression was verified in wildtype and heterozygous mouse eye and colon epithelia by immunohistochemistry. No histochemical reaction product was observed in nasal tissues, and there was no difference in olfactory function in knock-out mice compared to wildtype or heterozygous littermates.
We have successfully constructed a knock-out mouse model for mouse best-2. No obvious phenotype was detected. Similar to the Bestrophin (best-1) knock-out, the absence of best-2 appears to be well tolerated. Best-2 mRNA and protein expression appears to be predominantly confined to eye and colon epithelia as confirmed by X-gal staining and immunohistochemistry. Characterization of subtle phenotypes is ongoing.
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