Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.
Drug-inducible synergistic gene silencing with multiple small hairpin RNA molecules for gene function study in animal model.
复制标题
多个小发夹 RNA 分子的药物诱导协同基因沉默，用于动物模型中的基因功能研究。
DOI：
10.1007/s11248-014-9841-9
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发表时间：
2015-04
影响因子：
3
通讯作者：
Liu, Xiaoqing
中科院分区：
文献类型：
--
作者：
Ying, Ming;Chen, Guangfeng;Qiu, Yu;Shi, Xiujuan;Zhang, Chen;Wang, Qiuke;Yang, Shuzhang;Lu, Lixia;Yuan, Qionglan;Xu, Guotong;Jin, Zibing;Wu, Qiang;Liu, Xiaoqing
基因打靶是构建疾病模型的关键工具。然而，传统同源重组介导的基因敲除技术的应用由于缺乏快速频率保证的靶向方法而受到限制。尽管传统的小发夹 RNA (shRNA) 介导的基因沉默为基因靶向提供了一种替代方案，但与基因敲除相比，其应用经常因 RNA 干扰的表达效率较低而受到影响。在这里，我们提供了一种有效的基因靶向策略，涉及多个 shRNA 分子的药物诱导协同沉默。诱导后，所有测试的稳定转基因哺乳动物细胞系（包括 HEK293 和携带 shRNA 沉默盒的胚胎干细胞衍生的后代）中靶蛋白的水平均降至不可检测的水平。在携带针对视紫红质基因的沉默盒的转基因小鼠模型中，短时间诱导剂治疗足以消除视网膜中的视紫红质蛋白，导致与视紫红质突变小鼠中观察到的相似的视网膜表型变化。因此，从广泛的角度来看，这种诱导型 shRNA 基因靶向策略提供了一种与传统 RNA 干扰方法相当的真正的基因敲除替代方案。本文的在线版本 (doi:10.1007/s11248-014-9841-9) 包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1007/s11248-014-9841-9) contains supplementary material, which is available to authorized users.
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DOI：
10.1073/pnas.0800940105
发表时间：
2008-04-15
影响因子：
11.1
作者：
Santiago, Yolanda;Chan, Edmond;Collingwood, Trevor N.
通讯作者：
Collingwood, Trevor N.
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
48
作者：
Gaj, Thomas;Guo, Jing;Kato, Yoshio;Sirk, Shannon J.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III