Wadhwa Renu, Yaguchi Tomoko, Kaur Kamaljit, Suyama Eigo, Kawasaki Hiroyuki, Taira Kazunari, Kaul Sunil C
Gene Function Research Center, National Institute of Advanced Industrial Science & Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
J Biol Chem. 2004 Dec 3;279(49):51622-9. doi: 10.1074/jbc.M407428200. Epub 2004 Sep 24.
We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.
我们已采用基于杂交锤头状核酶的基因发现系统，通过体外成肌细胞分化试验来鉴定参与肌肉分化的功能基因。主要的肌肉调节基因（MyoD1、Mylk、肌球蛋白、肌细胞生成素和Myf5）被鉴定出来，证实了该方法的有效性。其他基因靶点包括肿瘤抑制因子和细胞周期调节因子（p19ARF和p21WAF1）、FGFR-4、纤连蛋白、Prkg2、Pdk4、fem以及六种新蛋白质。通过使用核酶和小干扰RNA特异性敲低已鉴定的三个靶点，证实了它们在成肌细胞分化中的功能参与。除了展示一种简单有效的分离参与肌肉分化的基因功能的方法外，我们首次报道，性别决定蛋白家族成员Fem的过表达导致肌管形成加速，而对其靶向作用则延缓了成肌细胞分化。这种功能性基因筛选不仅有助于理解肌肉分化的分子途径，还有助于设计针对肌病治疗的分子策略。
