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RNA interference pathway genes as tools for targeted genetic interference

a technology of rna interference pathway and gene, applied in the field of genes, can solve the problems of more undesirable effects of “cosuppression” and transgene silencing, and achieve the effects of promoting dsrna-mediated genetic interference, reducing or increasing the activity of the rnai pathway

Inactive Publication Date: 2005-05-12
UNIV OF MASSACHUSETTS MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023] An RNAi pathway component is a protein or nucleic acid that is involved in promoting dsRNA-mediated genetic interference. A nucleic acid component can be an RNA or DNA molecule. A mutation in a gene encoding an RNAi pathway component may decrease or increase RNAi pathway activity.

Problems solved by technology

Furthermore, the even more undesirable effect of “cosuppression” can occur in which a corresponding endogenous copy of the transgene becomes inactivated.
In some cases, transgene silencing leads to problems with the commercial or therapeutic application of transgenic technology to alter the genetic makeup of a cell, organism, or human patient.

Method used

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  • RNA interference pathway genes as tools for targeted genetic interference

Examples

Experimental program
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example 1

Strains and Alleles

[0127] The Bristol strain N2 was used as standard wild-type strain. The marker mutations and deficiencies used are listed by chromosomes as follows: LGI: dpy-14(e188), unc-13(e51); LGIII: dpy-17(e164), unc-32(e189); LGV: dpy-11(e224), unc-42(e270), daf-11(m87), eDf1, mDf3, nDf31, sDf29, sDf35, unc-76(e911). The C. elegans strain DP13 was used to generate hybrids for STS linkage-mapping (Williams et al., 1992, Genetics 131:609-624).

[0128] Sensitivity to RNAi was tested in the following strains. MT3126: mut-2(r459) (obtained from John Collins, Department of Biochemistry & Molecular Biology, University of New Hampshire, Durham, N. H.); dpy-19(n1347), TW410: mut-2([459) sem-4(n1378), NL917: mut-7(pk204), SS552: mes-2(bn76) rol-1(e91) / mnC1 (obtained from S. Strome, Biology Dept., Indiana University), SS449: mes-3(bn88) dpy-5(e61) (from S. Strome, supra); hDp20, SS268: dpy-11(e224) mes-4(bn23) unc-76(e91) / nT1, SS360: mes-6(bn66) dpy-20(e1282) / nT1, CB879: him-1 (e879)....

example 2

RNA Interference Assay

[0131] Genetic interference using RNAi administered by microinjection was performed as described in Fire et al., 1998, supra and Rocheleau et al., 1997, Cell 90:707-716. pos-1 cDNA clone yk61h1, par-2 cDNA clone yk96h7, sqt-3 cDNA clone yk75f2 were used to prepare dsRNA in vitro. These cDNA clones were obtained from the C. elegans cDNA project (Y. Kohara, Gene Network Lab, National Institute of Genetics, Mishima 411, Japan).

[0132] Genetic interference using RNAi administered by feeding was performed as described in Timmons and Fire, 1998, Nature 395:854. pos-1 cDNA was cloned into a plasmid that contains two T7 promoter sequences arranged in head-to-head configuration. The plasmid was transformed into an E. coli strain, BL21 (DE3), and the transformed bacteria were seeded on NGM (nematode growth medium) plates containing 60 μg / ml ampicillin and 80 μg / ml IPTG. The bacteria were grown overnight at room temperature to induce pos-1 dsRNA. Seeded plates (BL21(DE3)...

example 3

Identification of RNAi-Deficient Mutants

[0134] A method of screening for mutants defective in the RNAi pathway was devised that would permit the large-scale application of dsRNA to mutagenized populations. Feeding worms E. coli which express a dsRNA, or simply soaking worms in dsRNA solution, are both sufficient to induce interference in C. elegans (Timmons and Fire, 1998, supra; Tabara et al., 1998, Science 282:430-431). To carry out a selection, the feeding method was optimized to deliver interfering RNA for an essential gene, pos-1. C. elegans hermaphrodites that ingest bacteria expressing dsRNA corresponding to a segment of pos-1 are themselves unaffected but produce dead embryos with the distinctive pos-1 embryonic lethal phenotype.

[0135] To identify strains defective in the RNAi pathway, wild-type animals were mutagenized, backcrossed, and the F2 generation examined for rare individuals that were able to produce complete broods of viable progeny. Chemical mutagenesis was use...

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Abstract

Genes involved in double-stranded RNA interference (RNAi pathway genes) are identified and used to investigate the RNAi pathway. The genes and their products are also useful for modulating RNAi pathway activity.

Description

RELATED APPLICATION INFORMATION [0001] This application claims priority from provisional application Ser. No. 60 / 159,776, filed Oct. 15, 1999, and 60 / 193,218, filed Mar. 30, 2000.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH [0002] Funding for the work described herein was provided by the federal government (GM58800 and GM37706), which has certain rights in the invention.FIELD OF THE INVENTION [0003] This invention relates to the discovery of genes whose expression products are involved in mediation of genetic interference. BACKGROUND OF THE INVENTION [0004] All eukaryotic organisms share similar mechanisms for information transfer from DNA to RNA to protein. RNA interference represents an efficient mechanism for inactivating this transfer process for a specific targeted gene. Targeting is mediated by the sequence of the RNA molecule introduced to the cell. Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function (genetic interference) in several organisms in...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K38/00A61K48/00A61P43/00C07K14/435C07K16/18C12N5/10C12N15/67
CPCC12N15/67C07K14/43545A61P43/00
Inventor MELLO, CRAIGFIRE, ANDREWTABARA, HIROAKIGRISHOK, ALLA
Owner UNIV OF MASSACHUSETTS MEDICAL
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