Nucleic Acids Research, 2003, Vol. 31, No. 21 e127
© 2003 Oxford University Press
Inducible shRNA expression for application in a prostate cancer mouse model
Atugen AG, Otto Warburg Haus (No. 80), Robert-Roessle-Strasse 10, 13125 Berlin, Germany
*To whom correspondence should be addressed. Tel: +49 30 9489 2833; Fax: +49 30 9489 2801; Email: kaufmann{at}atugen.com
RNA interference (RNAi) is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression post-transcriptionally. Synthetic short interfering RNAs (siRNAs) as well as vector-based siRNA expression systems have been used successfully to silence gene expression in a variety of biological systems. We describe the development of an inducible siRNA expression system that is based on the tetracycline repressor and eukaryotic RNA polymerase III promoters (U6 and 7SK). For proof of concept we selectively inhibited expression of two catalytic subunits of the phosphatidylinositol 3-kinase (PI 3-kinase), p110
and p110ß, by using vector-derived short hairpin RNAs (shRNAs). Stable pools of human prostate cancer cells (PC-3) exhibiting reduced levels of both PI 3-kinase catalytic subunits due to the expression of corresponding shRNAs in an inducible fashion were established and analyzed for their invasive potential in vitro as well as in an orthotopic metastatic mouse model. This inducible system for RNAi allows an unbiased and comparable analysis of loss-of-function phenotypes by comparing selected isogenic cell populations on the induced and non-induced level. In addition, conditional RNAi allows the study of essential and multifunctional genes involved in complex biological processes by preventing inhibitory and compensatory effects caused by constitutive knockdown.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Zhang, C. Wang, N. Ke, J. Bliesath, J. Chionis, Q. S. He, Q.-x. Li, J. E. Chatterton, F. Wong-Staal, and D. Zhou A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals RNA, August 1, 2007; 13(8): 1375 - 1383. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Henriksen, C. Lokke, M. Hammero, D. Geerts, R. Versteeg, T. Flaegstad, and C. Einvik Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines Nucleic Acids Res., May 14, 2007; 35(9): e67 - e67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. De Paula, M. V. L.B. Bentley, and R. I. Mahato Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting RNA, April 1, 2007; 13(4): 431 - 456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Seibler, A. Kleinridders, B. Kuter-Luks, S. Niehaves, J. C. Bruning, and F. Schwenk Reversible gene knockdown in mice using a tight, inducible shRNA expression system Nucleic Acids Res., April 1, 2007; 35(7): e54 - e54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kappel, Y. Matthess, B. Zimmer, M. Kaufmann, and K. Strebhardt Tumor inhibition by genomically integrated inducible RNAi-cassettes Nucleic Acids Res., September 11, 2006; 34(16): 4527 - 4536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Lamarcq, B. J. Scherer, M. L. Phelan, N. N. Kalnine, Y. H. Nguyen, T. Kabakova, X. Chen, M. Tan, C. Chang, C. Berlon, et al. Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference J Biomol Screen, April 1, 2006; 11(3): 236 - 246. [Abstract] [PDF]
|
|
|
|
|
|
L. Amar, M. Desclaux, N. Faucon-Biguet, J. Mallet, and R. Vogel Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter Nucleic Acids Res., March 6, 2006; 34(5): e37 - e37. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Li, X. Lin, M. Staver, A. Shoemaker, D. Semizarov, S. W. Fesik, and Y. Shen Evaluating Hypoxia-Inducible Factor-1{alpha} as a Cancer Therapeutic Target via Inducible RNA Interference In vivo Cancer Res., August 15, 2005; 65(16): 7249 - 7258. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Coumoul, V. Shukla, C. Li, R.-H. Wang, and C.-X. Deng Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference Nucleic Acids Res., June 24, 2005; 33(11): e102 - e102. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Maeda, K. Fukushima, K. Nishizaki, and R. J.H. Smith In vitro and in vivo suppression of GJB2 expression by RNA interference Hum. Mol. Genet., June 15, 2005; 14(12): 1641 - 1650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Lelievre, P.-M. Bourbon, L.-J. Duan, R. L. Nussbaum, and G.-H. Fong Deficiency in the p110{alpha} subunit of PI3K results in diminished Tie2 expression and Tie2-/--like vascular defects in mice Blood, May 15, 2005; 105(10): 3935 - 3938. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Mittal Screening for Effective siRNAs and Their Inducible Expression in Mammalian Cells Am. Assoc. Cancer Res. Educ. Book, April 1, 2005; 2005(1): 357 - 361. [Full Text] [PDF]
|
|
|
|
|
|
S. D. Buckingham, B. Esmaeili, M. Wood, and D. B. Sattelle RNA interference: from model organisms towards therapy for neural and neuromuscular disorders Hum. Mol. Genet., October 1, 2004; 13(suppl_2): R275 - R288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Tondera, A. Santel, R. Schwarzer, S. Dames, K. Giese, A. Klippel, and J. Kaufmann Knockdown of MTP18, a Novel Phosphatidylinositol 3-Kinase-dependent Protein, Affects Mitochondrial Morphology and Induces Apoptosis J. Biol. Chem., July 23, 2004; 279(30): 31544 - 31555. [Abstract] [Full Text] [PDF]
|
|