Abstract
RNA interference (RNAi) is now a popular method for silencing gene expression in a variety of systems. RNAi methods use double-stranded RNAs (dsRNAs) to target complementary RNAs for destruction. In mammalian systems, very short dsRNAs (22–25 bp) such as short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are used to avoid endogenous nonspecific antiviral responses that target longer dsRNAs. siRNAs elicit a transient silencing response, while shRNAs can be expressed continuously to establish stable gene silencing. shRNAs can be introduced into cells and animals using a variety of standard vectors as well as retroviral or lentiviral expression systems. This chapter describes the design, construction, validation, and use of shRNAs for silencing genes. We report our results from testing a variety of shRNA design features and shRNA expression vectors. We also provide methods that use shRNAs to permit different levels of gene expression. Additionally, we discuss some aspects important for constructing an information pipeline to support development of a large shRNA library.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.
Baglioni, C. and Nilsen, T. W. (1983) Mechanisms of antiviral action of interferon. Interferon 5, 23–42.
Williams, B. R. (1997) Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem. Soc. Trans. 25, 509–513.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958.
Paddison, P. J., Caudy, A. A., and Hannon, G. J. (2002) Stable suppression of gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 1443–1448.
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.
Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508.
Sui, G., Soohoo, C., {mnAffar el}, B., Gay, F., Shi, Y., and Forrester, W. C. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–5520.
Yu, J. Y., DeRuiter, S. L., and Turner, D. L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–6052.
Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002) Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1–20.
Paddison, P. J. and Hannon, G. J. (2002) RNA interference: the new somatic cell genetics? Cancer Cell 2, 17–23.
Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon-Cardo, C., Hannon, G. J., and Lowe, S. W. (2003) An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33, 396–400.
McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L., and Kay, M. A. (2003) Inhibition of hepatitis B virus in mice by RNA interference. Nat. Biotechnol. 6, 639–644.
Carmell, M. A., Zhang, L., Conklin, D. S., Hannon, G. J., and Rosenquist, T. A. (2003) Germline transmission of RNAi in mice. Nat. Struct. Biol. 10, 91–92.
Semizarov, D., Frost, L., Sarthy, A., Kroeger, P., Halbert, D. N., and Fesik, S. W. (2003) Specificity of short interfering RNA determined through gene expression signatures. Proc. Natl. Acad. Sci. USA 100, 6347–6352.
Chi, J. T., Chang, H. Y., Wang, N. N., Chang, D. S., Dunphy, N., and Brown, P. O. (2003) Genomewide view of gene silencing by small interfering RNAs. Proc. Natl. Acad. Sci. USA 100, 6343–6346.
Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G., and Linsley, P. S. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637.
Singh, R. and Reddy, R. (1989) Gamma-monomethyl phosphate: a cap structure in spliceosomal U6 small nuclear RNA. Proc. Natl. Acad. Sci. USA 86, 8280–8283.
Zhang, H., Kolb, F. A., Brondani, V., Billy, E., and Filipowicz, W. (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415.
Kisielow, M., Kleiner, S., Nagasawa, M., Faisal, A., and Nagamine, Y. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA. Biochem. J. 363, 1–5.
Miller, V. M., **a, H., Marrs, G. L., Gouvion, C. M., Lee, G., Davidson, B. L., and Paulson, H. L. (2003) Allele-specific silencing of dominant disease genes. Proc. Natl. Acad. Sci. USA 100, 7195–7200.
Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243–247.
Lassus, P., Rodriguez, J., and Lazebnik, Y. (2002) Confirming specificity of RNAi in mammalian cells. Sci. STKE 147, PL13.
Lassus, P., Opitz-Araya, X., and Lazebnik, Y. (2002) Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 297, 1352–1354.
Forler, D., Kocher, T., Rode, M., Gentzel, M., Izaurralde, E., and Wilm, M. (2003) An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21, 89–92.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Paddison, P.J., Caudy, A.A., Sachidanandam, R., Hannon, G.J. (2004). Short Hairpin Activated Gene Silencing in Mammalian Cells. In: Gott, J.M. (eds) RNA Interference, Editing, and Modification. Methods in Molecular Biology, vol 265. Humana Press. https://doi.org/10.1385/1-59259-775-0:085
Download citation
DOI: https://doi.org/10.1385/1-59259-775-0:085
Publisher Name: Humana Press
Print ISBN: 978-1-58829-242-1
Online ISBN: 978-1-59259-775-8
eBook Packages: Springer Protocols