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10 - Six methods of inducing RNAi in mammalian cells

Published online by Cambridge University Press:  31 July 2009

Kathy Latham
Affiliation:
Ambion, Inc.
Vince Pallotta
Affiliation:
Ambion, Inc.
Lance Ford
Affiliation:
Ambion, Inc.
Mike Byrom
Affiliation:
Ambion, Inc.
Mehdi Banan
Affiliation:
Ambion, Inc.
Po-Tsan Ku
Affiliation:
Ambion, Inc.
David Brown
Affiliation:
Ambion, Inc.
Krishnarao Appasani
Affiliation:
GeneExpression Systems, Inc., Massachusetts
Andrew Fire
Affiliation:
Stanford University, California
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Summary

Using siRNAs to silence gene expression

The capacity to utilize a cell's naturally occurring RNA interference (RNAi) pathway to silence target gene expression has precipitated a new era in functional genomic research (McManus and Sharp, 2003; Dillin, 2003). RNAi can be induced in mammalian cells by small interfering RNAs (siRNAs) that target complementary mRNAs for degradation. Because of its specificity, reproducibility, and ease of use, RNAi is greatly accelerating the functional characterization of disease-relevant genes for drug discovery, target validation, and basic research efforts.

There are six basic methods for generating siRNAs. siRNAs can be prepared in vitro by chemical synthesis, in vitro transcription, or RNAse III/Dicer digestion of long double-stranded RNAs (dsRNAs). The in vitro prepared siRNAs can then be delivered to mammalian cells by a variety of methods including lipofection and electroporation. Alternatively, siRNAs can be expressed in mammalian cells from DNA plasmids, viral vectors, or PCR products bearing an siRNA template adjacent to a compatible promoter. As with the in vitro prepared siRNAs, DNA plasmids and PCR products can be delivered to mammalian cells by a variety of methods – methods such as transfection agents and electroporation. Viral vectors, on the other hand, are first packaged into viral particles that are then used to infect cells.

Various methods for inducing RNAi in mammalian cells are described in the following sections. The relative advantages and disadvantages for each method are discussed. In addition, design criteria for both siRNAs and siRNA expression templates are described.

Type
Chapter
Information
RNA Interference Technology
From Basic Science to Drug Development
, pp. 147 - 160
Publisher: Cambridge University Press
Print publication year: 2005

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References

Ashrafi, K., Chang, F. Y., Watts, J. L., Fraser, A. G., Kamath, R. S., Ahringer, J. and Ruvkun, G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature, 421, 268–272CrossRefGoogle ScholarPubMed
Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell, 101, 235–238CrossRefGoogle ScholarPubMed
Brummelkamp, T. R., Bernards, R. and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553CrossRefGoogle ScholarPubMed
Calegari, F., Haubensak, W., Yang, D., Huttner, W. B. and Buchholz, F. (2002). Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proceedings of the National Academy of Sciences USA, 99, 14236–14240CrossRefGoogle ScholarPubMed
Castanotto, D., Li, H. and Rossi, J. J. (2002). Functional siRNA expression from transfected PCR products. RNA, 8, 1454–1460CrossRefGoogle ScholarPubMed
Devroe, E. and Silver, P. A. (2002). Retrovirus-delivered siRNA. BMC Biotechnology, 2(1), 15CrossRefGoogle ScholarPubMed
Dillin, A. (2003). The specifics of small interfering RNA specificity. Proceedings of the National Academy of Sciences USA, 100, 6289–6291CrossRefGoogle ScholarPubMed
Donzé, O. and Picard, D. (2002). RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Research, 30(10), e46CrossRefGoogle ScholarPubMed
Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W. and Tuschl, T. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. European Molecular Biology Organization Journal, 20, 6877–6888CrossRefGoogle ScholarPubMed
Ilves, H., Barske., C., Junker., U., Bohnlein, E. and Veres, G. (1996). Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: A comparative study. Gene, 171, 203–208CrossRefGoogle ScholarPubMed
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. Nature Genetics, 33, 396–400CrossRefGoogle ScholarPubMed
Jacque, J.-M., Triques, K. and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature, 418, 435–438CrossRefGoogle ScholarPubMed
Kay, M. A., Glorioso, J. C. and Naldini, L. (2001). Viral vectors for gene therapy: The art of turning infectious agents into vehicles of therapeutics. Nature Medicine, 7(1), 33–40CrossRefGoogle ScholarPubMed
Kunath, T., Gish, G., Lickert, H., Jones, N., Pawson, T. and Rossant, J. (2003). Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nature Biotechnology, 21, 559–561CrossRefGoogle ScholarPubMed
Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J., Ehsani, A., Salvaterra., P. and Rossi, J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnology, 19, 500–505CrossRefGoogle Scholar
Lee, S. S., Lee, R. Y., Fraser, A. G., Kamath, R. S., Ahringer, J. and Ruvkun, G. A. (2003). Systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nature Genetics, 33, 40–48CrossRefGoogle ScholarPubMed
Matsuguchi, T., Masuda, A., Sugimoto, K., Nagai, Y. and Yoshikai, Y. (2003). JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation. European Molecular Biology Organization Journal, 22, 4455–4464CrossRefGoogle ScholarPubMed
Matsukura, S., Jones, P. A. and Takai, D. (2003) Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Research, 31(15), e77CrossRefGoogle ScholarPubMed
McManus, M. T. and Sharp, P. A. (2003). Gene silencing in mammals by small interfering RNAs. Nature Medicine, 3, 737–747Google Scholar
Miller, V. M., Xia, H., Marrs, G. L., Gouvion, C. M., Lee, G., Davidson, B. L. and Paulson, H. L. (2003). Allele-specific silencing of dominant disease genes. Proceedings of the National Academy of Sciences USA, 100, 7195–7200CrossRefGoogle ScholarPubMed
Miyagishi, M. and Taira, K. (2002). U6-promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnology, 19, 497–500CrossRefGoogle Scholar
Muda, M., Worby, C. A., Simonson-Leff, N., Clemens, J. C. and Dixon, J. E. (2002). Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochemical Journal, 366, 73–77CrossRefGoogle ScholarPubMed
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 & Development, 16, 948–958CrossRefGoogle ScholarPubMed
Paul, C. P., Good, P. D., Winer, I. and Engelke, D. R. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnology, 19, 505–508CrossRefGoogle Scholar
Pothof, J., Haaften, G., Thijssen, K., Kamath, R. S., Fraser, A. G., Ahringer, J., Plasterk, R. H. A. and Tijsterman, M. (2003). Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes & Development, 17, 443–448CrossRefGoogle Scholar
Qin, X-F., An, D. S., Chen, I. S. Y. and Baltimore, D. (2003). Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proceedings of the National Academy of Sciences USA, 100, 183–188CrossRefGoogle Scholar
Robinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Zhang, M., McManus, M. T., Gertler, F. B., Scott, M. L. and Parijs, L. V. (2003). A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nature Genetics, 33, 401–406CrossRefGoogle Scholar
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. Proceedings of the National Academy of Sciences USA, 100, 6347–6352CrossRefGoogle ScholarPubMed
Shang, Y. and Brown, M. (2002). Molecular Determinants for the Tissue Specificity of SERMs. Science 295, 2465–2468CrossRefGoogle ScholarPubMed
Sharp, P. A. (2001). RNA interference – 2001. Genes & Development, 15, 485–490CrossRefGoogle ScholarPubMed
Sui, G., Soohoo, C., Affar, E.-B., Gay, F., Shi, Y., Forrester, W. C. and Shi, Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proceedings of the National Academy of Sciences USA, 99, 5515–5520CrossRefGoogle ScholarPubMed
Trotta, R., Vignudelli, T., Candini, O., Intine, R. V., Pecorari, L., Guerzoni, C., Santilli, G., Byrom, M. W., Goldoni, S., Ford, L. P., Caligiuri, M. A., Maraia, R. J., Perrotti, D. and Calabretta, B. (2003). BCR/ABL activates mdm2 mRNA translation via the La antigen. Cancer Cell, 3, 145–160CrossRefGoogle ScholarPubMed
Wetering, M., Oving, I., Muncan, V., Fong, M. T. P., Brantjes, H., Leenen, D., Holstege, F. C. P., Brummelkamp, T. R., Agami, R. and Clevers, H. (2003). Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. European Molecular Biology Organization Reports, 4, 609–615Google ScholarPubMed
Vigna, E. and Naldini, L. (2000). Lentiviral vectors: Excellent tools for experimental gene transfer and promising candidates for gene therapy. The Journal of Gene Medicine, 2, 308–3163.0.CO;2-3>CrossRefGoogle ScholarPubMed
Xia, H., Paulson, H. L. and Davidson, B. L. (2002). siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnology, 20, 1006–1010CrossRefGoogle ScholarPubMed
Yang, D., Buchholz, F., Huang, Z., Goga, A., Chen, C-Y., Brodsky, F. M. and Bishop, M. J. (2002). Short RNA duplexes produced by hydrolysis with Escherichia coli RNAse III mediate effective RNA interference in mammalian cells. Proceedings of the National Academy of Sciences USA, 99, 9942–9947CrossRefGoogle ScholarPubMed
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. Proceedings of the National Academy of Sciences USA, 99, 6047–6052CrossRefGoogle ScholarPubMed

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