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Y. Shi, S. Bassnett; Inducible Gene Expression In The Mouse Lens . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2553.
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To evaluate the utility of the Cre–ERTM system for inducing transgene expression in the lens.
We obtained two strains of mice. One strain (Cre–ERTM) carried Cre–recombinase fused to the mutated ligand–binding domain of the human estrogen receptor. The mutation ensures that the receptor binds tamoxifen (TM; a synthetic estrogen) but not native estrogen. The reporter strain (Z/EG) carried a lacZ transgene flanked by loxP sites. In the presence of cre–recombinase, the lacZ gene is excised and the expression of a downstream green fluorescent protein (GFP) gene is permitted. The expression of both transgenes was under the control of a ß–actin promoter. Crosses were established between Cre–ERTM mice and Z/EG mice. Progeny carrying both transgenes were injected intraperitonealy with tamoxifen (2 mg) at p21, and the subsequent expression of GFP was assessed by western blotting and confocal microscopy.
Western blot analysis indicated that tissues varied widely in their expression of GFP prior to tamoxifen injection. Heart tissue, for example, had high baseline GFP expression, whereas, in the lens, GFP expression was undetectable. After TM injection there was a large increase in GFP expression in all tissues. In the lens epithelium, GFP–positive cells were evident five days after tamoxifen injection. Expressing cells were highly fluorescent, although the number of GFP–positive cells was relatively low (<5%). GFP–positive fiber cells were not observed initially, but were evident a few days later, presumably as a result of the differentiation of GFP–positive epithelial cells.
In the present study we evaluated the utility of the Cre–ERTM inducible expression system in the lens. Our data suggest that this approach will be useful for inactivating genes in individual, identified, lens epithelial cells. For applications that require the induced expression of cre–recombinase in a higher proportion of lens cells, it may be necessary to drive Cre–ERTM expression with a strong, lens–specific promoter.
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