Gene targeting is a critical tool for construction\nof disease models. However, the application\nof traditional homologous recombination-mediated\ngene knockout technology is limited by the absence\nof rapid frequency-guaranteed targeting methods.\nAlthough conventional small hairpin RNA (shRNA)-\nmediated gene silencing offers an alternative for gene\ntargeting, its application is frequently compromised by\nlower expression efficiency via RNA interference\ncompared to gene knockout. Here we provide an\nefficient gene targeting strategy involving drug-inducible\nsynergistic silencing with multiple shRNA molecules.\nOn induction, the levels of the target proteins\ndecreased to undetectable levels in all the tested stable\ntransgenic mammalian cell lines, including HEK293\nand embryonic stem cell-derived progenies carrying\nshRNA silencing cassettes. In a transgenic mouse\nmodel carrying a silencing cassette targeting the\nrhodopsin gene, short-time inducer treatment was\nsufficient to ablate the rhodopsin protein in the retina,\nresulting in similar retinal phenotypic changes as those\nobserved in rhodopsin mutant mice. Therefore, on a\nbroad basis, this inducible shRNA gene targeting\nstrategy offers a true gene knockout alternative comparable\nto conventional RNA interference approaches.
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