Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model

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• 作者：Ming Ying (1) (2)
  Guangfeng Chen (1)
  Yu Qiu (1)
  Xiujuan Shi (1)
  Chen Zhang (1)
  Qiuke Wang (1)
  Shuzhang Yang (1)
  Lixia Lu (1)
  Qionglan Yuan (1)
  Guotong Xu (1)
  Zibing Jin (3)
  Qiang Wu (4)
  Xiaoqing Liu (1) (2)
  
  1. Shanghai Tenth People鈥檚 Hospital ; Tongji University School of Medicine ; 301 Yanchangzhong Rd. ; Shanghai ; 200072 ; China
  2. Shenzhen Key Laboratory of Marine Bioresources and Ecology ; College of Life Sciences ; Shenzhen University ; 3688 Nanhai Ave. ; Shenzhen ; 518060 ; China
  3. Division of Ophthalmic Genetics ; The Eye Hospital of Wenzhou Medical College and Lab for Stem Cell and Retinal Regeneration ; School of Ophthalmology and Optometry ; Wenzhou Medical College ; No. 270 ; West Xueyuan Rd. ; Wenzhou ; 325027 ; China
  4. Department of Ophthalmology ; Affiliated Sixth People鈥檚 Hospital ; Shanghai Jiao Tong University ; No. 600 Yishan Rd. ; Shanghai ; 200233 ; China
  
• 关键词：RNAi ; shRNA ; Gene targeting ; Gene silencing ; Drug ; inducible
• 刊名：Transgenic Research
• 出版年：2015
• 出版时间：April 2015
• 年：2015
• 卷：24
• 期：2
• 页码：309-317
• 全文大小：2,971 KB
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• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Biomedicine
  Molecular Medicine
  Plant Genetics and Genomics
  Animal Genetics and Genomics
  Plant Sciences
  Human Genetics
  
• 出版者：Springer Netherlands
• ISSN：1573-9368

文摘

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.