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Nucleic Acids Research, 2003, Vol. 31, No. 15 e77
© 2003 Oxford University Press

Establishment of conditional vectors for hairpin siRNA knockdowns

Shiro Matsukura, Peter A. Jones* and Daiya Takai

Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA

*To whom correspondence should be addressed. Tel: +1 323 865 0816; Fax: +1 323 865 0102; Email: jones_p{at}ccnt.hsc.usc.edu
Present addresses:
Shiro Matsukura, Department of Surgery, Saga Medical School, 5-1-1, Nabeshima, Saga, Japan
Daiya Takai, Department of Respiratory Medicine, Graduate School of Medicine and Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyoku, Tokyo, Japan

Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.


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