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RNA Interference in Caenorhabditis Elegans

Darryl Conte¹, Craig C. Mello¹

¹University of Massachusetts Medical School, Worcester, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 26.3

DOI:

10.1002/0471142727.mb2603s62

Online Posting Date:

May, 2003

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Abstract

RNAi has become an essential tool in C. elegans research. Procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA and soaking in dsRNA solution are described in this unit.

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Unit Introduction
Basic Protocol 1: Microinjecting Worms with dsRNA
Alternate Protocol: Microinjection into unc-42 rde-1 Animals Crossed to N2 Males to Examine Zygotic RNAi Phenotypes
Basic Protocol 2: Feeding Worms dsRNA
Basic Protocol 3: Soaking Worms in dsRNA
Support Protocol 1: Picking and Propagating C. Elegans
Support Protocol 2: Sexing and Mating C. Elegans
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Microinjecting Worms with dsRNA

Materials

0.1 to 1 µg/µl dsRNA
Halocarbon oil Series 700 (Halocarbon Products)
L4 to young-adult staged C. elegans (e.g., strain N2; CGC)
M9 buffer (see recipe)
NGM plates seeded with E. coli (see recipe)

Loading capillary (see recipe)
Microinjection needle (see recipe)
Inverted microscope with micromanipulator and microinjection assembly with N₂ gas supply (e.g., TRITECH Research Microinjector System)
Agarose microinjection pads (see recipe)
30-mm Petri dish lid
Dissecting microscope
Platinum wire pick flattened at the end

Additional reagents and materials for transferring and propagating C. elegans (see Support Protocol 1)

Alternate Protocol: Microinjection into unc-42 rde-1 Animals Crossed to N2 Males to Examine Zygotic RNAi Phenotypes

Additional Materials (also see Basic Protocol 1)

Male N2 C. elegans (CGC)
unc-42 rde-1 C. elegans (WM36; CGC)
Mating plates (see recipe)
Additional reagents and equipment for transferring and propagating (see Support Protocol 1), and mating C. elegans

Basic Protocol 2: Feeding Worms dsRNA

Materials

cDNA
L4440 double T7 RNAi feeding vector (Fig. 26.3.5; A. Fire, http://www.ciwemb.edu)
E. coli RNAi feeding strain HT115 (CGC)
Terrific broth (TB; UNIT 1.1) containing 50 µg/ml each ampicillin and tetracycline (UNIT 1.4)
TB containing 50 µg/ml ampicillin
M9 buffer (see recipe)
M9/15% glycerol (see recipe)
NGM/amp/IPTG plates (see recipe)
Gravid/young adult, L1 larvae, or L4 larvae C. elegans of appropriate strain(s) (e.g., N2, CGC)
NGM/amp/IPTG plates (see recipe) seeded with HT115 containing empty L4440 RNAi feeding vector

Additional reagents and equipment for subcloning DNA fragments (UNIT 3.16), transforming E. coli (UNIT 1.8), growing E. coli (UNIT 1.2), and transferring and propagating C. elegans (see Support Protocol 1)

NOTE: Perform each RNAi experiment in triplicate.

Basic Protocol 3: Soaking Worms in dsRNA

Materials

dsRNA
Nonspecific dsRNA—i.e., dsRNA that is nonhomologous to any C. elegans sequence and which is comparable in length to the experimental dsRNA (e.g., gfp; optional)
Soaking solution (see recipe)
C. elegans (e.g., N2, CGC)
M9 buffer (see recipe)
NGM plates (see recipe) with and without seeded E. coli OP50 (see recipe)

65°C water bath
0.2- and 2.0-ml microcentrifuge tubes
5-ml glass pipet

Additional reagents and equipment for ethanol precipitation (UNIT 2.1A)

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Figures

Figure 26.3.1

Loading the microinjection needle.

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Figure 26.3.2

Attaching the microinjection needle to the microinjection assembly via tubing.

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Figure 26.3.3

Overview of the microinjection technique. (A) The injection needle enters the field of view from the left of the image and is oriented above and roughly parallel to the body axis of the worm. (B to D) The needle is lowered until the tip touches the worm forming a slight indentation. The worm is pushed against the needle until the tip of the needle penetrates the cuticle. (E to G) The dsRNA solution is injected into the body cavity of the worm and flows in both directions away from the tip (indicated by the bars and arrows). (H) The needle is then backed out of the worm by moving the worm away from the needle.

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Figure 26.3.4

Schematic of the zygotic RNAi procedure.

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Figure 26.3.5

Multiple cloning site of the double T7 RNAi feeding vector L4440. Opposing phage T7 promoters flank the multiple cloning site of L4440. The restriction sites shown are present exclusively in the MCS with the unique sites indicated in bold.

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Literature Cited

Literature Cited
	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 421:268-272.
	Bei, Y., Hogan, J., Berkowitz, L.A., Soto, M., Rocheleau, C.E., Pang, K.M., Collins, J., and Mello, C.C. 2002. SRC-1 and Wnt signaling act together to specify endoderm and to control cleavage orientation in early C. elegans embryos. Dev. Cell 3:113-125.
	Bosher, J.M. and Labouesse, M. 2000. RNA interference: Genetic wand and genetic watchdog. Nat. Cell. Biol. 2:E31-36.
	The C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: A platform for investigating biology. Science. 282:2012-2018.
	Dudley, N.R., Labbe, J.C., and Goldstein, B. 2002. Using RNA interference to identify genes required for RNA interference. Proc. Natl. Acad. Sci. U.S.A. 99:4191-4196.
	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.
	Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
	Fraser, A.G., Kamath, R.S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M., and Ahringer, J. 2000. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408:325-330.
	Gonczy, P., Echeverri, C., Oegema, K., Coulson, A., Jones, S.J., Copley, R.R., Duperon, J., Oegema, J., Brehm, M., Cassin, E., Hannak, E., Kirkham, M., Pichler, S., Flohrs, K., Goessen, A., Leidel, S., Alleaume, A.M., Martin, C., Ozlu, N., Bork, P., and Hyman, A.A. 2000. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408:331-336.
	Grishok, A., Tabara, H., and Mello, C.C. 2000. Genetic requirements for inheritance of RNAi in C. elegans. Science 287:2494-2497.
	Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D.L., Fire, A., Ruvkun, G., and Mello, C.C. 2001. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23-34.
	Hannon, G.J. 2002. RNA interference. Nature 418:244-251.
	Hope, I.A., Ed. 1999. C. elegans: A Practical Approach. The Practical Approach Series. Oxford University Press, New York.
	Jansen, G., Hazendonk, E., Thijssen, K.L., and Plasterk, R.H. 1997. Reverse genetics by chemical mutagenesis in Caenorhabditis elegans. Nat. Genet. 17:119-121.
	Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., Ahringer, J. 2001. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2:RESEARCH0002.
	Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D.P., Zipperlen, P., and Ahringer, J. 2003. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421:231-237. [comment 220-221]
	Liu, L.X., Spoerke, J.M., Mulligan, E.L., Chen, J., Reardon, B., Westlund, B., Sun, L., Abel, K., Armstrong, B., Hardiman, G., King, J., McCague, L., Basson, M., Clover, R., and Johnson, C.D. 1999. High-throughput isolation of Caenorhabditis elegans deletion mutants. Genome Res. 9:859-867.
	Maeda, I., Kohara, Y., Yamamoto, M., and Sugimoto, A. 2001. Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Curr. Biol. 11:171-176.
	Mello, C. and Fire, A. 1995. DNA transformation. Methods Cell. Biol. 48:451-482.
	Montgomery, M.K., Xu, S., and Fire, A. 1998. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 95:15502-1557.
	Piano, F., Schetter, A.J., Mangone, M., Stein, L., and Kemphues, K.J. 2000. RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr. Biol. 10:1619-1622.
	Riddle, D.L., Blumenthal, T., Meyer, B.J., Priess, J.R. 1997. C. ELEGANS II. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
	Tabara, H., Grishok, A., and Mello, C.C. 1998. RNAi in C. elegans: Soaking in the genome sequence. Science 282:430-431.
	Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and Mello, C.C. 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123-132.
	Tavernarakis, N., Wang, S.L., Dorovkov, M., Ryazanov, A., and Driscoll, M. 2000. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24:180-183.
	Timmons, L., Court, D.L., and Fire, A. 2001. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:103-112.
	Wood, W.B. (ed.) 1988. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Key References
	Hope, 1999. See above.
A very good source for general information on culturing and maintaining worms as well as a comprehensive microinjection protocol for germline transformation.
	Kamath et al., 2001. See above.
This manuscript provides an in depth analysis of the RNAi by feeding technique as well as a comparison to RNAi by microinjection.
	Riddle et al., 1997. See above.
C. ELEGANS II provides an extensive overview of worm biology.
	Wood, 1988. See above.
The predecessor of C. ELEGANS II (Riddle et al., 1997). The Nematode is full of important information about the biology of C. elegans.
Internet Resources
	http://www.wormbase.org
Wormbase is an essential tool for C. elegans biologists, and provides an abundance of useful information regarding the genome and biology of C. elegans. The curators at Wormbase have provided annotations of each of the predicted genes including RNAi phenotypes and expression data as well as links to useful sites. The curators are constantly updating and upgrading the site.
	http://nematode.lab.nig.ac.jp/db/rnai_s/RNAiBySoaking.html
This site includes several protocols for RNAi by soaking as well as for preparing template DNA and dsRNA for the RNAi by soaking protocols.
	http://www.rnai.org
The RNAi database is a searchable database of ovary or germline-enriched genes by gene or by phenotype.
	http://c.elegans.tripod.com/RNAi.htm
The RNAi@elegans.net page has many informative links for RNAi in C. elegans and other organisms.
	http://elegans.swmed.edu
The C. elegans WWW server.
	http://nematode.lab.nig.ac.jp/db/index.html
NEXTDB is an expression pattern database based on in situ hybridizations using expressed sequence tags.