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J. Biol. Chem., Vol. 279, Issue 49, 51622-51629, December 3, 2004

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Use of a Randomized Hybrid Ribozyme Library for Identification of Genes Involved in Muscle Differentiation^*

Renu Wadhwa, Tomoko Yaguchi, Kamaljit Kaur, Eigo Suyama, Hiroyuki Kawasaki, Kazunari Taira, and Sunil C. Kaul¶

From the Gene Function Research Center, National Institute of Advanced Industrial Science & Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan and the Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan

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 (p19^ARF and p21^WAF1), 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.

Received for publication, July 2, 2004 , and in revised form, September 24, 2004.

^* This work was supported in part by a research grant from the National Institute of Advanced Industrial Science & Technology (AIST). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Gene Function Research Center, AIST, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Tel.: 81-29-861-6713; Fax: 81-29-861-2900; E-mail: s-kaul{at}aist.go.jp.

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