Abstract
RNA interference (RNAi) has become a powerful tool for the specific silencing of gene transcription. Especially the targeting of genes in mammalian cells has been greatly improved by generating plasmid-based and viral vector-based systems. This permits expression of short hairpin RNA (shRNA) on a longterm basis. However, and inducible expression of shRNA is required, if the target is essential for cell survival. We developed a doxycycline-inducible two-plasmid system for the expression of a ribozyme-processed shRNA. In contrast to other existing systems, we use the highly specific T7 phage RNA polymerase, which does not interact with cellular factors; therefore, interference with cellular functions is limited. One plasmid is responsible for doxycline-dependent expression of T7 RNA polymerase and a second plasmid expresses a ribozyme-processed shRNA under the control of a T7 promoter. Our results showed that doxycycline-dependent expression of T7 RNA polymerase was tightly controlled and expression of an shRNA against firefly luciferase inhibited 86% of luciferase activity. In conclusion, our plasmid system provides a very useful tool for analyzing essential gene functions in vitro.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bantounas, I., Phylactou, L. A., and Uney, J. B. (2004). RNA interference and the use of small interferting RNA to study gene function in mammalian systems. J. Mol. Endocrinol., 33, 545–557.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1988) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.
Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 101, 25–33.
Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev, 15, 188–200.
Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcir, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Abbas-Terki, T., Blanco-Bose, W., Deglon, N., Pralong, W., and Aebischer, P. (2002) Lentiviral-mediated RNA interference. Hum. Gene Ther. 13, 2197–2201.
Barton, G. M., and Medzhitov, R. (2002) Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. USA 99, 14,943–14,945.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553.
Tiscornia, G., Singer, O., Ikawa, M., and Verma, I. M. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100, 1844–1848.
Tomar, R. S., Matta, H., and Chaudhary, P. M. (2003) Use of adeno-associated viral vector for delivery of small intefering RNA. Oncogene 22, 5712–5715
Gupta, S., Schoer, R. A., Egan, J. E., Hannon, G. J., and Mittal, V. (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1927–1932.
Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, M., and Tuschl, T. (2001) Functional anatomy of siRNA for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20, 6877–6888.
van de Wetering, M., Oving, I., Muncan, V., et al., (2003) Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615.
Tunitskaya, V. L. and Kochetkov, S. N. (2002) Structural-functional analysis of bacteriophage T7 RNA polymerase. Biochemistry (Mosc.) 67, 1124–1135.
Luo, G. X., Luytjes, W., Enami, M. and Palese, P. (1991) The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure. J. Virol, 65, 2861–2867.
Perrotta, A. T. and Been, M. D. (1991) A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature, 350, 434–436.
McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., and Sharp, P. A. (2002) Gene silencing using micro-RNA designed hairpins. RNA 8, 842–850.
Ahn, J., Urist, M., and Prives, C. (2003) Questioning the role of checkpoint kinase 2 in the p53 DNA damage response. J. Biol. Chem. 278, 20,480–20,489.
Verma, U. N., Surabhi, R. M., Schmaltieg, A., Becerra, C., and Gaynor, R. B. (2003) Small interfering RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Clin. Cancer Res. 9, 1291–1300.
Novina, C. D., Murray, M. F., Dykxhoorn, D. M., et al. (2002) siRNA-directed inhibition of HIV-1 infection. Nat. Med., 8, 681–686.
McManus, M. T. and Sharp, P. A. (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3, 737–747.
Author information
Authors and Affiliations
Corresponding author
Additional information
These authors contributed equally to the work.
Rights and permissions
About this article
Cite this article
Hamdorf, M., Muckenfuss, H., Tschulena, U. et al. An inducible T7 RNA polymerase-dependent plasmid system for the expression of short hairpin RNAs. Mol Biotechnol 33, 13–21 (2006). https://doi.org/10.1385/MB:33:1:13
Issue Date:
DOI: https://doi.org/10.1385/MB:33:1:13