Six methods of inducing RNAi in mammalian cells

doi:10.1017/CBO9780511546402.013

10 - Six methods of inducing RNAi in mammalian cells

Published online by Cambridge University Press: 31 July 2009

Kathy Latham ,

Vince Pallotta ,

Lance Ford ,

Mike Byrom ,

Mehdi Banan ,

Po-Tsan Ku and

David Brown

Edited by

Krishnarao Appasani

Foreword by

Andrew Fire and

Marshall Nirenberg

Show author details

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

Book contents

Get access

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

DOI: https://doi.org/10.1017/CBO9780511546402.013 [Opens in a new window]

Publisher: Cambridge University Press

Print publication year: 2005

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Book purchase

Temporarily unavailable

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, 268–272CrossRef Google Scholar PubMed

Bass, B. L. (2000). Double-stranded RNA as a template for gene silencing. Cell, 101, 235–238CrossRef Google Scholar PubMed

Brummelkamp, T. R., Bernards, R. and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553CrossRef Google Scholar PubMed

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, 14236–14240CrossRef Google Scholar PubMed

Castanotto, D., Li, H. and Rossi, J. J. (2002). Functional siRNA expression from transfected PCR products. RNA, 8, 1454–1460CrossRef Google Scholar PubMed

Devroe, E. and Silver, P. A. (2002). Retrovirus-delivered siRNA. BMC Biotechnology, 2(1), 15CrossRef Google Scholar PubMed

Dillin, A. (2003). The specifics of small interfering RNA specificity. Proceedings of the National Academy of Sciences USA, 100, 6289–6291CrossRef Google Scholar PubMed

Donzé, O. and Picard, D. (2002). RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Research, 30(10), e46CrossRef Google Scholar PubMed

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, 6877–6888CrossRef Google Scholar PubMed

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, 203–208CrossRef Google Scholar PubMed

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, 396–400CrossRef Google Scholar PubMed

Jacque, J.-M., Triques, K. and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature, 418, 435–438CrossRef Google Scholar PubMed

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), 33–40CrossRef Google Scholar PubMed

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, 559–561CrossRef Google Scholar PubMed

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, 500–505CrossRef Google 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, 40–48CrossRef Google Scholar PubMed

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, 4455–4464CrossRef Google Scholar PubMed

Matsukura, S., Jones, P. A. and Takai, D. (2003) Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Research, 31(15), e77CrossRef Google Scholar PubMed

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, 7195–7200CrossRef Google Scholar PubMed

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, 497–500CrossRef Google 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, 73–77CrossRef Google Scholar PubMed

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, 948–958CrossRef Google Scholar PubMed

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, 505–508CrossRef Google 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, 443–448CrossRef Google 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, 183–188CrossRef Google 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, 401–406CrossRef Google 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, 6347–6352CrossRef Google Scholar PubMed

Shang, Y. and Brown, M. (2002). Molecular Determinants for the Tissue Specificity of SERMs. Science 295, 2465–2468CrossRef Google Scholar PubMed

Sharp, P. A. (2001). RNA interference – 2001. Genes & Development, 15, 485–490CrossRef Google Scholar PubMed

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, 5515–5520CrossRef Google Scholar PubMed

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, 145–160CrossRef Google Scholar PubMed

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 Scholar PubMed

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>CrossRef Google Scholar PubMed

Xia, H., Paulson, H. L. and Davidson, B. L. (2002). siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnology, 20, 1006–1010CrossRef Google Scholar PubMed

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, 9942–9947CrossRef Google Scholar PubMed

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, 6047–6052CrossRef Google Scholar PubMed