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Gene Silencing by RNAi in Mammalian Cells

Matthias John¹, Anke Geick¹, Philipp Hadwiger¹, Hans‐Peter Vornlocher¹, Olaf Heidenreich²

¹Ribopharma AG, Kulmbach, Germany
²University of Tübingen, Tübingen, Germany

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 26.2

DOI:

10.1002/0471142727.mb2602s62

Online Posting Date:

May, 2003

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Abstract

This unit provides information how to use short interfering RNA (siRNA) for sequence specific gene silencing in mammalian cells. Several ways for siRNA generation and optimisation, as well as recommendations for cell transfection are presented.

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Unit Introduction
Basic Protocol: Liposome-Mediated Transfection of Mammalian Cells with siRNA
Alternate Protocol: Electroporation of Mammalian Cells with siRNA
Support Protocol 1: Annealing Equimolar siRNA
Support Protocol 2: Liposome-Mediated Cotransfection of Reporter Genes with siRNA
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: Liposome-Mediated Transfection of Mammalian Cells with siRNA

Materials

Mammalian cells to be transfected (e.g., HeLa-S3)
Complete medium (APPENDIX 3F)
20 µM annealed siRNA (see Support Protocol 1)
Serum-free medium (APPENDIX 3F)
Lipofectamine PLUS (Invitrogen)

12-well culture dishes (2-cm diameter)
1.5-ml polypropylene tubes

Additional reagents and equipment for mammalian cell tissue culture, including trypsinization (APPENDIX 3F)

NOTE: Lipofectamine PLUS is used in this protocol as it works well for HeLa-3 cells. Other transfection reagents can also be used by following manufacturer's instructions. Lipofectamine PLUS consists of two reagents, the transfection reagent itself (Lipofectamine) and an enhancer (PLUS).

Alternate Protocol: Electroporation of Mammalian Cells with siRNA

Additional Materials (also see Basic Protocol)

4-mm electroporation cuvettes (e.g., Equibio ECU104)
Square-wave electroporator (e.g., Fischer EPI 2500)
Culture vessel

Support Protocol 1: Annealing Equimolar siRNA

Materials

siRNA sense and antisense strands (see Critical Parameters)
Annealing buffer: 100 mM NaCl in 20 mM sodium phosphate buffer, pH 6.8 (see APPENDIX 2 for buffer)

Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (UNIT 2.7) and ethidium bromide staining (UNIT 4.9)

Support Protocol 2: Liposome-Mediated Cotransfection of Reporter Genes with siRNA

Additional Materials (also see Basic Protocol)

Reference plasmid expressing -galactosidase (e.g., pCMV beta, Clontech)
Plasmid expressing reporter gene fusion (e.g. HCV-Luciferase in pcDNA3.1+, Invitrogen)
TE buffer (APPENDIX 2)
5 µM annealed siRNA (see Support Protocol 1)
GenePORTER 2 kit (Gene Therapy Systems):
DNA diluent B
GenePORTER 2 Reagent (liposome transfection reagent)
Annealing buffer: 100 mM NaCl in 20 mM sodium phosphate buffer, pH 6.8 (see APPENDIX 2 for buffer)
96-well culture dishes

Additional reagents and equipment for -galactosidase and firefly luciferase assays (UNIT 9.7B)

Looking for materials? Suppliers Guide

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Figures

Figure 26.2.1

A model for RNA interference in mammalian cells. Long double-stranded RNAs are processed by dicer. RISC induces a target-site-specific cleavage of RNA using siRNA and dicer products as a guide. See text for more details.

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Figure 26.2.2

Examples of synthetic siRNA and their target gene. Efficient siRNA have double-stranded regions of 19 to 21 nucleotides and 2-nucleotide overhangs at the 3¢ end on both strands. A substitution of ribouridine bases by deoxythymidine at the 3¢ prime ends may stabilize the siRNA in solution, but is not necessary for efficient gene silencing in mammalian cells. The two targets of the siRNA shown at the top of the figure are lamin A/C (Elbashir et al., 2001) and firefly luciferase (Hohjoh,2002), respectively. The two siRNAs shown at the bottom of the figure both have chloramphenicol acetyl transferase (Caplen et al., 2002) as their target.

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Figure 26.2.3

Model for plasmid-derived synthesis of siRNA in vivo. An RNA polymerase III promoter drives the expression of short hairpin-like RNA with a proposed 19-nucleotide duplex. The complementary sense and antisense sequence of the final siRNA are linked by a loop of 8 to 10 nucleotides (A). This hairpin RNA is probably subject to intracellular processing. The remaining double-stranded molecules (B) possess sequence homology with the target gene and perform like functional siRNA (Brummelkamp et al., 2002).

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Figure 26.2.4

A recombinant fusion gene serves as a reporter system for siRNA efficiency in mammalian cells. A 26-nucleotide hepatitis C virus sequence is placed upstream of the firefly luciferase open reading frame. The viral sequence possesses the ability to form a stem-loop structure. Two siRNAs were designed to target the mRNA. HCV1+2 (A) is directed against the hepatitis C region, whereas LUC1+2 (B) is targeting the open reading frame of the fusion gene. The siRNA C1+2 (C) is not related to the reporter mRNA and serves as a negative control.

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Figure 26.2.5

In liver cells, the reporter gene is expressed at a 5-fold lower level in presence of HCV1+2 and LUC1+2 compared to control siRNA or absence of siRNA. Note the slight stimulation of reporter gene activity by C1+2. Refer to Figure 26.2.4 for siRNA sequences.

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Literature Cited

Literature Cited
	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.
	Caplen, N.J., Parrish, S., Imani, F., Fire, A., and Morgan, R.A. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U.S.A. 98:9742-9747.
	Chiu, Y.-L. and Rana, T.M. 2002. RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol. Cell. 10:549-561.
	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. 2001 Nature. 411:494-498.
	Hannon, G.J. 2002. RNA interference. Nature 418:244-251.
	Heidenreich, O., Krauter, J., Riehle, H., Hadwiger, P., John, M., Heil, G., Vornlocher, HP., and Nordheim, A. 2002. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells. Blood: In press. [epub ahead of print]
	Hohjoh, H. 2002. RNA interference (RNAi) induction with various types of synthetic oligonucleotide duplexes in cultured human cells. FEBS Lett. 521:195-199.
	Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E., and Prydz, H. 2002. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucl. Acids. Res. 30:1757-1766.
	Kolykhalov, A. A., Feinstone, S.M., and Rice, C.M. 1996. Identification of a highly conserved sequence element at the 3¢ terminus of hepatitis C virus genome RNA. J. Virol. 70:3363-3371.
	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 transcript in human cells. Nat. Biotechnol. 20:500-505.
	MacManus, M.T. and Sharp, P.A. 2002. Gene silencing in mammals by small interfering RNAs. Nature Genet. 3:737-747.
	Martinez, J., Patkaniowska, A., Urlaub, H., Lührmann, R., and Tuschl, T. 2002. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 110:563-574.
	Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S.-K., Collman, R.G., Lieberman, J., Shankar, P., and Sharp, P.A. 2002. SiRNA-directed inhibition of HIV infection. Nature Med. 8:681-686.
	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.
	Schwarz, D.S. and Zamore, P.D. 2002. Why do miRNAs live in the miRNP Genes Dev. 16:1025-1031.
	Sharp, P.A. 2001. RNA interference–2001. Genes Dev. 15:485-490.
	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. Proc. Natl. Acad. Sci. U.S.A. 99:5515-5520.
	Zamore, P.D. RNA interference: Listening to the sound of silence. 2001. Nat. Struct. Biol. 8:746-750.
	Zeng, Y. and Cullen, B.R. 2002. RNA interference in human cells is restricted to the cytoplasm. RNA. 8:855-860.
Key References
	Elbashir, S.M., Harborth, J., Weber, K., and Tuschl, T. 2002. Analysis of gene function in somatic cells using small interfering RNAs. Methods. 26:199-213.
The pioneers in mammalian RNA interference present a concise description of planning and performing siRNA experiments in this reference.
Internet Resources
	http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html
This site provides suggestions and recommendations for experiments with siRNA.
	http://www.ambion.com
Web site with plenty of background information about RNA interference and regular literature updates.