CRISPR/Cas9-mediated knockout of Lim-domain only four retards organ of Corti cell growth.
CRISPR/Cas9-mediated knockout of Lim-domain only four retards organ of Corti cell growth.
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DOI：
10.1002/jcb.26529
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发表时间：
2018-04
影响因子：
4
通讯作者：
Jamesdaniel S
中科院分区：
文献类型：
--
作者：
Rathinam R;Rosati R;Jamesdaniel S
Lim-domain only 4（LMO 4）在介导高效抗癌药物顺铂的耳毒性副作用中起关键作用。然而，耳蜗LMO 4介导耳病理学的信号传导机制尚未完全理解。敲除细胞培养模型是研究新基因的功能作用和描绘相关信号通路的有用工具。因此，通过使用CRISPR（成簇的规则散布的短回文重复序列）/Cas9（CRISPR相关蛋白9）系统产生LMO 4敲除的Corti器官细胞。UB/OC 1细胞中LMO 4的成功敲除通过免疫印迹中不存在LMO 4蛋白条带来验证。尽管LMO 4的敲除延缓了细胞的生长速率和迁移潜力，但它并没有抑制它们的长期活力，因为LMO 4敲除的UB/OC 1细胞能够存活、增殖和形成集落。此外，LMO 4的敲除并没有改变毛细胞的生物标志物肌球蛋白VIIa的表达，这表明敲除细胞保留了耳蜗感觉受体细胞的重要特征。因此，这项研究的结果表明，CRISPR/Cas9系统是一种简单而通用的敲除Corti细胞器官中感兴趣基因的方法，LMO 4敲除UB/OC 1细胞是研究LMO 4在耳毒性中功能作用的可行实验模型。
Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system. Successful knockout of LMO4 in UB/OC1 cells was verified by the absence of LMO4 protein bands in immunoblots. Though the Knockout of LMO4 retarded the growth rate and the migratory potential of the cells it did not inhibit their long-term viability as the LMO4 knockout UB/OC1 cells were able to survive, proliferate, and form colonies. In addition, the knockout of LMO4 did not alter the expression of myosin VIIa, a biomarker of hair cells, suggesting that the knockout cells retain important characteristic features of cochlear sensory receptor cells. Thus, the findings of this study indicate that CRISPR/Cas9 system is a simple and versatile method for knocking out genes of interest in organ of Corti cells and that LMO4 knockout UB/OC1 cells are viable experimental models for studying the functional role of LMO4 in ototoxicity.
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影响因子：
4.4
作者：
Riccardi S;Bergling S;Sigoillot F;Beibel M;Werner A;Leighton-Davies J;Knehr J;Bouwmeester T;Parker CN;Roma G;Kinzel B
通讯作者：
Kinzel B
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
5.3
作者：
Hahm, KM;Sum, EYM;Orkin, SH
通讯作者：
Orkin, SH
影响因子：
8
作者：
Lu, Z.;Lam, K. S.;Andersen, B.
通讯作者：
Andersen, B.
影响因子：
5.3
作者：
Schock, Sarah C.;Xu, Jin;Chen, Hsiao-Huei
通讯作者：
Chen, Hsiao-Huei