title: Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.
abstract: Gene targeting is a critical tool for construction of disease models. However, the application of traditional homologous recombination-mediated gene knockout technology is limited by the absence of rapid frequency-guaranteed targeting methods. Although conventional small hairpin RNA (shRNA)-mediated gene silencing offers an alternative for gene targeting, its application is frequently compromised by lower expression efficiency via RNA interference compared to gene knockout. Here we provide an efficient gene targeting strategy involving drug-inducible synergistic silencing with multiple shRNA molecules. On induction, the levels of the target proteins decreased to undetectable levels in all the tested stable transgenic mammalian cell lines, including HEK293 and embryonic stem cell-derived progenies carrying shRNA silencing cassettes. In a transgenic mouse model carrying a silencing cassette targeting the rhodopsin gene, short-time inducer treatment was sufficient to ablate the rhodopsin protein in the retina, resulting in similar retinal phenotypic changes as those observed in rhodopsin mutant mice. Therefore, on a broad basis, this inducible shRNA gene targeting strategy offers a true gene knockout alternative comparable to conventional RNA interference approaches.
authors.affiliation: Shanghai Tenth People's Hospital, Tongji University School of Medicine, 301 Yanchangzhong Rd., Shanghai, 200072, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Ying
authors.forename: Guangfeng
authors.initials: G
authors.lastname: Chen
authors.forename: Yu
authors.initials: Y
authors.lastname: Qiu
authors.forename: Xiujuan
authors.initials: X
authors.lastname: Shi
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.forename: Qiuke
authors.initials: Q
authors.lastname: Wang
authors.forename: Shuzhang
authors.initials: S
authors.lastname: Yang
authors.forename: Lixia
authors.initials: L
authors.lastname: Lu
authors.forename: Qionglan
authors.initials: Q
authors.lastname: Yuan
authors.forename: Guotong
authors.initials: G
authors.lastname: Xu
authors.forename: Zibing
authors.initials: Z
authors.lastname: Jin
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wu
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D034741:RNA, Small Interfering / Q000235:genetics*; D012243:Rhodopsin / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D012243:Rhodopsin