WO2001048183A2 - Inhibition d'arn a double brin - Google Patents

Inhibition d'arn a double brin

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Publication number
WO2001048183A2
WO2001048183A2 PCT/EP2000/013149 EP0013149W WO0148183A2 WO 2001048183 A2 WO2001048183 A2 WO 2001048183A2 EP 0013149 W EP0013149 W EP 0013149W WO 0148183 A2 WO0148183 A2 WO 0148183A2
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WIPO (PCT)
Prior art keywords
nematode
strain
elegans
double
promoters
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PCT/EP2000/013149
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English (en)
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WO2001048183A3 (fr
Inventor
Geert Plaetinck
Katherine Mortier
Ann Lissens
Thierry Bogaert
Original Assignee
Devgen Nv
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Application filed by Devgen Nv filed Critical Devgen Nv
Priority to AU21715/01A priority Critical patent/AU2171501A/en
Publication of WO2001048183A2 publication Critical patent/WO2001048183A2/fr
Publication of WO2001048183A3 publication Critical patent/WO2001048183A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis

Definitions

  • the present invention is concerned with ways of improving the efficiency of double stranded RNA inhibition as a method of inhibiting gene expression in nematode worms such as C. elegans .
  • the invention relates to the finding that the susceptibility of nematode worms such as C. elegans to double stranded RNA inhibition is affected by changes in the genetic background of the worms.
  • the present inventors have utilized the double stranded RNA inhibition technique and applied it further to devise novel and inventive methods of (i) assigning functions to genes or DNA fragments which have been sequenced in various projects, such as, for example, the human genome project and which have yet to be accorded a particular function, and (ii) identifying DNA responsible for conferring a particular phenotype.
  • Such methods are described in the applicant's co-pending application number WO 00/01846.
  • Processes for introducing RNA into a living cell, either In vivo or ex vivo, in order to inhibit expression of a target gene in that cell are additionally described in WO 99/32619.
  • RNA interference in vivo .
  • One of the most straightforward approaches is simple injection of double-stranded RNA into a body cavity.
  • a more elegant solution is to feed the nematodes on food organisms, generally bacteria, which express a double stranded RNA of the appropriate sequence, corresponding to a region of the target gene.
  • RNA interference in nematodes has now determined that the phenomenon of RNA interference in nematodes ' following ingestion of food organisms capable of expressing double-stranded RNA is dependent both on the nature of the food organism and on the genetic background of the nematodes themselves. These findings may be exploited to provided improved methods of double-stranded RNA inhibition.
  • a method of inhibiting expression of a target gene in a nematode worm comprising feeding to said nematode worm a food organism which is capable of producing a double- stranded RNA structure having a nucleotide sequence substantially identical to a portion of said target gene following ingestion of the food organism by the nematode, wherein the nematode has a non wild-type genetic background selected to provide increased sensitivity to RNA interference as compared to wild type.
  • Caenorhabd.i t is elegans is the preferred nematode worm for use in the method of the invention although the method could be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus Caenorhabdi tis .
  • the term "microscopic" nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform mid- to high- throughput screening.
  • the nematode has a non wild-type genetic background which confers greater sensitivity to RNA interference phenomena (abbreviated herein to RNAi) as compared to the equivalent wild type nematodes.
  • RNAi RNA interference phenomena
  • introduction of double-stranded RNA (abbreviated herein to dsRNA) into a non wild-type strain according to the invention results in greater inhibition of expression of the target gene.
  • this greater level of inhibition may be detectable at the phenotypic level as a more pronounced phenotype.
  • the nematode having non wild-type genetic background may, advantageously, be a mutant strain.
  • Mutations which have the effect of increasing susceptibility of the nematode to RNAi may, for example, affect the stability of dsRNA or the kinetics of dsRNA turnover within cells of the worm or the rate of uptake of dsRNA synthesised by a food organism.
  • Suitable mutant strains include mutant strains exhibiting knock-out or loss-of-function mutations in one or more genes encoding proteins involved in RNA synthesis, RNA degradation or the regulation of these processes .
  • the nematode is a mutant strain, more preferably a mutant C. elegans, which exhibits reduced activity of one or more nucleases compared to wild-type.
  • Suitable strains include mutant strains exhibiting knock-out or loss- of-function mutations in one or more genes encoding nucleases, such as RNases.
  • a particularly preferred example is the nuc-1 strain. This mutant C. elegans strain is known per se in the art.
  • the nematode is a mutant strain, more preferably a mutant C. elegans , which exhibits increased gut uptake compared to wild- type.
  • Particularly preferred examples of such strains are the so-called C. elegans gun mutants described herein.
  • the nematode may be a transgenic worm comprising one or more transgenes which increase gut uptake relative to wild- type.
  • increased gut uptake is taken to mean increased uptake of foreign particles from the gut lumen and may encompass both increased gut permeability and increased gut molecular transport compared to wild-type C. elegans .
  • C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up liquid and subsequently spitting it out is called pharyngeal pumping. Once the food particles have been internalised via pharyngeal pumping their contents must cross the gut itself in order to reach target sites in the worm. There are multiple factors which effect the uptake of compounds from the gut lumen to the surrounding tissues. These include the action of multi-drug resistance proteins, multi-drug resistance related proteins and the P450 cytochromes as well as other enzymes and mechanisms available for transport of molecules through the gut wall.
  • C. elegans mutants which exhibit increased uptake of foreign molecules through the gut may be obtained from the C. elegans mutant collection at the C. elegans Genetic Center, University of Minnesota, St Paul, Minnesota, or may be generated by standard methods. Such methods are described by Anderson in Methods in Cell Biology, Vol 48, C. elegans : Modern biological analysis of an organism" Pages 31 to 58. Several selection rounds of the PCR technique can be performed to select a mutant worm with a deletion in a desired gene. Alternatively, a population of worms could be subjected to random mutagenesis and worms exhibiting the desired characteristic of increased gut uptake selected using a phenotypic screen, such as the dye uptake method described herein.
  • transgenic worms may be generated with the appropriate characteristics.
  • Methods of preparing transgenic worms are well known in the art and are particularly described by Craig Mello and Andrew Fire, Methods in Cell Biology, Vol 48, Ed. H.F. Epstein and D.C. Shakes, Academic Press, pages 452-480.
  • Worms exhibiting the desired characteristics of increased gut uptake can be identified using a test devised by the inventors based on uptake of a marker precursor molecule which is cleaved by the action of enzymes present in the gut lumen to generate a marker molecule which produces a detectable signal, such as fluorescence.
  • a suitable marker precursor molecule is the- fluorescent dye precursor BCECF-AM available from Molecular Probes (Europe BV) , Netherlands.
  • This dye only becomes fluorescent when cleaved by esterases and maintained at a pH above 6.
  • the pH of the gut lumen is usually 5 or below.
  • any BCECF-AM taken up through the pharynx into the gut lumen is not fluorescent until cleaved and the cleaved portion has entered the cells surrounding the lumen which are at a higher pH.
  • this dye is able to quickly identify mutant or otherwise modified worms which have increased gut transport or permeability. There is a gradual increase in fluorescence in the tissues surrounding the gut while the gut lumen remains dark. The fluorescence can be detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
  • J. elegans gun mutant strain bg85 was deposited on 23 December 1999 at the BCCM/LMG " culture collection, Laboratorium Voor Microbiologie, ⁇ niversiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium under accession number LMBP 5334CB.
  • the bg85 mutation refers to the specific mutation (s) present in the bg85 strain which is/are responsible for conferring the gun phenotype . It is also within the scope of the invention to use a non wild-type nematode strain, preferable a C. elegans strain, having multiple mutations which affect sensitivity to RNAi.
  • a preferred type of multiple mutant is one having at least one mutation which results in reduced nuclease activity compared to wild type and at least one mutation which results in increased gut uptake compared to wild type.
  • An example of such a mutant is a C. elegans strain having the nuc-1 mutation and at least one further gun mutation.
  • double mutants having the nuc-1 mutation and a gun mutation exhibit enhanced sensitivity to RNAi as compared to either nuc-1 or gun single mutants.
  • the N2 strain can be obtained from the C. elegans Genetic Center, University of Minnesota, St Paul, Minnesota, USA.
  • the food organism for use in the above aspect of the invention is preferably a bacterium such as, for example, a strain of E. coli . It will, however, be appreciated that any other type of food organism on which nematodes feed and which is capable of producing dsRNA could be used.
  • the food organism may be genetically modified to express a double-stranded RNA of the appropriate sequence, as will be understood with reference to the examples included herein.
  • One convenient way in which this may be achieved in a bacterial food organism is by transforming the bacterium with a vector comprising a promoter or promoters positioned to drive transcription of a DNA sequence to RNA capable of forming a double-stranded structure. Examples of such vectors will be further described below.
  • the actual step of feeding the food organism to the nematode may be carried out according to procedures known in the art, see WO 00/01846.
  • the feeding of the food organisms to the nematodes is performed on standard agar plates commonly used for culturing C. elegans in the laboratory.
  • the step of feeding the food organism to the nematodes may also be carried out in liquid culture, for example in the wells of 96-well microtitre assay plates.
  • the inventors have further observed that variations in the food organism can result in enhanced in vivo RNAi when the food organism is ingested by a nematode worm.
  • the invention provides a method of inhibiting expression of a target gene in a nematode worm comprising feeding to said nematode worm a food organism capable of producing a double-stranded RNA structure having a nucleotide sequence substantially identical to a portion of said target gene following ingestion of the food organism by the nematode, wherein the food organism carries a modification selected to provide increased expression or persistence of the doubled-stranded RNA compared to a food organism which does not carry the modification.
  • the modification present in the food organism can be any modification which results in increased expression of the dsRNA or in increased persistence of the dsRNA.
  • Suitable modifications might include mutations within the bacterial chromosome which affect RNA stability and/or degradation or mutations which have a direct effect on the rate of transcription.
  • the food organism is an RNAse III minus E. coli strain, or any other RNAse negative strain.
  • a method of inhibiting expression of a target gene in a nematode worm comprising introduction of a DNA capable of producing a double-stranded RNA structure having a nucleotide sequence substantially identical to a portion of said target gene in said nematode, wherein the nematode is one which exhibits increased gut uptake compared to wild type.
  • nematodes which exhibit increase gut uptake as described herein also show increased uptake of DNA molecules capable of producing double-stranded RNA structures following ingestion into a nematode.
  • the DNA is in the form of a vector comprising a promoter or promoters orientated to relative to a sequence of DNA such that they are capable of driving transcription of the said DNA to make RNA capable of forming a double-stranded structure upon binding of an appropriate RNA polymerase to the promoter or promoters.
  • RNA duplex a DNA fragment corresponding to a region of the target gene is flanked by two opposable polymerase-specific promoters which are preferably identical. Transcription from the opposable promoters produces two complementary RNA strands which can anneal to form an RNA duplex.
  • the plasmid pGNl described herein is an example of a vector comprising two opposable T7 promoters flanking a multiple cloning site for insertion of a DNA fragment of the appropriate sequence, corresponding to a region of a target gene.
  • pGN8 is an example of a vector derived from pGNl containing a fragment of the C. elegans unc-22 gene.
  • DNA fragments corresponding to a region of the target gene may be placed both in the sense and the antisense orientation downstream of a single promoter.
  • the sense/antisense fragments are co-transcribed to generate a single RNA strand which is self- complementary and can therefore form an RNA duplex.
  • the polymerase-specific T3, T7 and SP6 promoters are useful for driving transcription of the RNA. Expression from these promoters is dependent on expression of the cognate polymerase.
  • the nematode itself may be adapted to express the appropriate polymerase. Expression of the polymerase may be general and constitutive, but could also be regulated under a tissue-specific promoter, an inducible promoter, a temporally regulated promoter or a promoter having a combination of such characteristics.
  • elegans strains harboring a transgene encoding the desired polymerase under the control of an appropriately-regulated promoter can be constructed according to methods known per se in the art and described, for example, by Craig Mello and Andrew Fire in Methods in Cell Biology, Vol 48, Ed. H. F. Epstein and D. C. Shakes, Academic Press, pp 452-480.
  • the advantage of adapting the nematode to express the required polymerase is that it is possible to control inhibition of expression of the target gene in a tissue-specific and/or temporally specific manner by placing expression of the polymerase under the control of an appropriately regulated promoter.
  • DNA into nematodes in accordance with the method of the invention can be achieved using a variety of techniques, for example by direct injection into a body cavity or by soaking the worms in a solution containing the DNA.
  • the DNA is in the form of a vector as described herein, e.g. a plasmid harboring a cloned DNA fragment between two flanking T7 promoters, then dsRNA corresponding to this DNA fragment will be formed in the nematode resulting in down regulation of the corresponding gene.
  • the introduced DNA can form an extrachromosomal array, which array might result in a more catalytic knock-out or reduction of function phenotype.
  • the DNA might also become integrated into the genome of the nematode, resulting in the same catalytic knock out or reduction of function phenotype, but which is stably transmittable .
  • the double- stranded RNA structure may be formed by two separate complementary RNA strands or a single self- complementary strand, as described above. Inhibition of target gene expression is sequence-specific in that ' only nucleotide sequences corresponding to the duplex region of the dsRNA structure are targeted for inhibition.
  • dsRNA comprising a nucleotide sequence identical to a portion of the target gene, although RNA sequences with minor variations such as insertions, deletions and single base substitutions may also be used and are effective for inhibition. It will be readily apparent that 100% sequence identity between the dsRNA and a portion of the target gene is not absolutely required for inhibition and the phrase "substantially identical" as used herein is to be interpreted accordingly.
  • sequences which are substantially identical will share at least 90%, preferably at least 95% and more preferably at least 98% nucleic acid sequence identity. Sequence identity may be conveniently calculated based on an optimal alignment, for example using the BLAST program accessible at WW . ncbi . nlm. ih . gov.
  • Figure 1 is a plasmid map of the vector pGNl containing opposable T7 promoters flanking a multiple cloning site and an ampicillin resistance marker.
  • Figure 2 is a plasmid map of the vector pGN8 (a genomic fragment of the C. elegans unc-22 gene cloned in pGNl) .
  • Figure 3 is a plasmid map of the vector pGN29 containing two T7 promoters and two T7 terminators flanking SstXI sites. This vector permits cloning of DNA fragments linked to BstXI adaptors.
  • Figure 4 is a plasmid map of the vector pGN39 containing two T7 promoters and two T7 terminators flanking attR recombination sites (based on the
  • GatewayTM cloning system of Life Technologies, Inc GatewayTM cloning system of Life Technologies, Inc.
  • Figure 5 is a plasmid map of the vector pGX22 (a fragment of the C. elegans gene C04H5.6 cloned in pGN29) .
  • Figure 6 is a plasmid map of the vector pGX52 (a fragment of the C. elegans gene KllD9.2b cloned in pGN29) .
  • Figure 1 is a plasmid map of the vector pGXl04 (a fragment of the C. elegans gene Y57G11C.15 cloned in pGN29) .
  • Figure 8 is a plasmid map of the vector pGZ8 (a fragment of the C. elegans gene T25G3.2 cloned in pGN3 ) .
  • Figure 9 shows the results of an RNAi experiment in which wild-type (N2) or nuc-1 strain C. elegans in liquid culture were fed with E. coli containing the plasmid pGX22.
  • Figure 10 shows the results of an RNAi experiment in which wild-type (N2) or nuc-1 strain C. elegans in liquid culture were fed with E. coli containing the plasmid pGX52.
  • Figure 11 shows the results of an RNAi experiment in which wild-type (N2) or nuc-1 strain C. elegans in liquid culture were fed with E . coli containing the plasmid pGXGZ ⁇ .
  • Figure 12 shows the results of an RNAi experiment in which wild-type (N2) or nuc-1 strain C. elegans in liquid culture were fed with E . coli containing the plasmid pGXl04
  • MC1061 F-araD139 ⁇ (ara -leu) 7696 galEl ⁇ galK16 ⁇ (lac) X74 rpsl (Str r ) hsdR2 (r m k + ) mcrA mcrBl regular host for various plasmids, - Wert an et al., (1986) Gene 49:253-262,
  • B21(DE3) F- ompT (lon) hsdS B (r B " ,m B ⁇ ; an E. coli B strain) with DE3, a ⁇ prophage carrying the T7 RNA polymerase gene. regular host for IPTG inducible T7 polymerase expression,
  • HT115 (DE3) : F- mcrA mcrb IN (rrnD-rrnE) 1 ⁇ - rncl 4 : : trl 0 (DE3 lysogen: lacUV5 promoter-T7polymerase) host for IPTG inducible T7 polymerase expression, - RNaselll-,
  • C. elegans nuc-1 (el393) : C. elegans strain with a reduced endonuclease activity (>95%) ; condensed chromatin persists after programmed cell death; ingested (bacterial) DNA in the intestinal lumen is not degraded.
  • el392 strong allele: has been used for the experiments described below
  • n887 resembles el392
  • n334 weaker allele
  • pGNl A vector encoding for ampicillin resistance, harbouring a multiple cloning site between two convergent T7 promoters.
  • pGN8 pGNl containing a genomic fragment of unc-22. Decreased unc-22 expression via RNAi results in a "twitching" phenotype in C. elegans .
  • 12-well micro-titer plates were filled with approximately 2 ml of NGM agar per well (1 litre of NGM agar: 15g Agar, lg peptone, 3g NaCl, 1ml cholesterol solution (5 mg/ml in EtOH) , with sterile addition after autoclaving of 9.5 ml 0.1M CaCl 2 , 9.5 ml 0.1 ml MgS0 4 , 25 ml IM KH 2 P0 4 /K 2 HP0 4 buffer pH 6 and 5 ml nystatin solution (dissolved 10 mg/ml in 1:1 EtOH:CH 3 COONH 4 7.5 M) .
  • the dried plates were spotted with approximately 50 ⁇ l of an overnight culture of bacteria.
  • IPTG induction 50 ⁇ l of a 10 mM stock solution of IPTG was dropped on top of the bacteria lawn, and incubated at 37°C for approximately 4 hours.
  • Individual nematodes at the L4 growth stage were then placed in single wells. In each well 4 nematodes, and the plates were further incubated at 20°C for 6 days to allow offspring to be formed.
  • the Fl offspring of the seeded nematodes were tested for the twitching phenotype.
  • E. coli MC1061 shows that no twitching could be observed in this experiment. Neither the N2 nematodes nor the nuc-1 nematodes showed any twitchers. This is to be expected as E. coli MC1061 does not produce any T7 RNA polymerase, and hence the unc-22 fragment cloned in pGN8 is not expressed as dsRNA.
  • RNAi effect of the unc-22 dsRNA was even more pronounced in C. elegans strain nuc-1 than in the wild type N2 strain.
  • the nuc-1 mutation results in the non-degradation or at least in a slower degradation of DNA, as the NUC-1 protein is known to be involved in DNAse activity, we clearly observe an enhancement of the RNAi induced phenotype in C. elegans with a nuc-1 background.
  • the nuc-1 mutation has not been cloned yet, but it has been described that the gene is involved in nuclease activity, and more particularly
  • NUC-1 protein DNAse activity. If the NUC-1 protein is a nuclease, it may also have activity on nuclease activity on dsRNA, which would explain the enhanced RNAi phenotype.
  • the nuc-1 gene product may be a nuclease, or a regulator of nuclease activity. As the mode of action of RNAi is still not understood, it is also possible that the NUC-1 protein is interfering in the mode of action of RNAi. This would explain why a nuc-1 mutant is more sensitive to RNAi.
  • RNA interference observed with the unc-22 dsRNA is even higher. In comparison with strain BL21(DE3) this could be expected, as HT115(DE3) is a RNAse III minus strain, and hence is expected to produce larger amounts of dsRNA, resulting in more prominent RNAi. This indicates further that the RNAi observed in this experiment is the result of the dsRNA produced by the bacteria fed to the C. elegans . Feeding C. elegans nuc-1 with HT115(DE3) harbouring pGN8 also results in higher RNA interference phenotype than feeding the same bacteria to C. elegans wild-type strain N2. Once ' again this indicates that improved RNAi can realised using a nuclease negative C. elegans and more particularly with a with the C. elegans nuc-1 (el392) strain.
  • RNA interference can be achieved in C. elegans by feeding the worms with bacteria that produce dsRNA.
  • the efficiency of this RNA interference is dependent both on the E. coli strain and on the genetic background of the C. elegans strain.
  • This can be realised by using efficient RNA expression systems such as T7 RNA polymerase and RNAase negative strains, such as RNaselll minus stains.
  • the level of dsRNA production varied: HT115 (DE3) >BL21 (DE3) >MC1061.
  • RNA interference is high in C. elegans strains that are nuclease negative, or that are influenced in their nuclease activity. This can be realised by using a mutant strain such as C. elegans nuc-1 . In this example the sensitivity to RNAi varied: C. elegans nuc-1 » C. elegans N2 Example 2
  • RNAi by feeding dsRNA producing bacteria in selected C. elegans strains-Comparison of the nuc-1 strain with several mutants which show improved gut uptake, (designated herein ⁇ gun' mutants) .
  • Strains bg77, bg78, bg83, bg84, bg85, bg86, bg87, bg88 and bg89 are typical gun mutant C. elegans strains isolated using selection for increased gut uptake (gun phenotype) with the marker dye BCECF-AM.
  • bacterial strain HT115(DE3) shows a better RNAi sensitivity than bacterial strain BL21(DE3) the nuc-1 C. elegans strain is a better strain than the Wild-type N2 strain for RNAi sensitivity - various gun mutants (improved gut uptake mutants) and more particularly the gun mutant strains bg77, bg84, bg85, bg86 show improved sensitivity to RNAi compared to Wild-type .
  • a double mutant C. elegans strain shows even greater sensitivity to RNAi compared to wild-type:
  • Double mutants were constructed to test the prediction that gun/nuc mutants would even show more enhanced RNAi sensitivity.
  • the crossing strategy with gun strain bg85 is shown, similar crosses can be conducted with other gun strains, such as bg77, bg84 and bg86.
  • F2 cross nuc-1 x gun(bg85)/+; nuc-1/0 males (50%) nuc-1 x +/+; nuc-1/0 males (50%)
  • F3 single gun(bg85)/+; nuc-1 hermaphrodites (25%) +/+; nuc-1 hermaphrodites (75%)
  • F4 single gun(bg85); nuc-1 (1/4 of every 4th plate high staining with BCECF)
  • F5 retest gun(bg85); nuc-1 (100% progeny of F4 singled high staining with BCECF)
  • the fluorescence precursor BCECF-AM is used (obtainable from Molecular probes) .
  • the precursor BCECF-AM is cleaved by esterases present in the gut of the worm to generate the dye BCECF which is fluorescent at pH values above 6. This allows selection for worms that have a gun phenotype.
  • BCECF-AM is taken up through the pharynx into the gut lumen and is not fluorescent until it has been cleaved, and the BCECF portion has entered the cells surrounding the lumen. Wild-type worms will show slower or no increase in BCECF fluorescence.
  • RNAi was performed in liquid culture instead of on agar plates. We show here for a number of genes that the RNAi effect is more penetrant using the nuc-1 strain than the N2 strain, and that RNAi can be performed in liquid.
  • C. elegans nuc-1 (el393) : C. elegans strain with a reduced endonuclease activity (>95%) ; condensed chromatin persists after programmed cell death; ingested (bacterial) DNA in the intestinal lumen is not degraded.
  • el392 strong allele: has been used for the experiments described below
  • n887 resembles el392
  • n334 weaker allele
  • pGX22 a vector encoding ampicillin resistance, containing a genomic fragment of cos id C04H5.6 corresponding to a member of the RNA helicase family.
  • pGX52 a vector encoding ampicillin resistance, containing a genomic fragment of cosmid KllD9.2b corresponding to sarco/endoplasmic Ca2 + ATPase also known as SERCA.
  • pGZ18 a vector encoding ampicillin resistance, containing a genomic fragment of cosmid T25G3.2 corresponding to a chitin like synthase gene.
  • pGX104 a vector encoding ampicillin resistance, containing a genomic fragment of cosmid Y57G11C.15 corresponding to sec-61, a transport protein.
  • RNAi of this gene interferes with the generation of offspring.
  • SERCA a sarco/endoplasmic Ca 2+ ATPase.
  • a strong RNAi phenotype causes an acute lethal phenotype.
  • a less penetrant RNAi effect results in loss of offspring.
  • T25G3.2 a chitin like synthase gene.
  • RNAi of this gene causes dead eggs.
  • sec-61 a transport protein.
  • a strong RNAi phenotype causes an acute lethal phenotype. A less penetrant RNAi effect results in loss of offspring.
  • - RNAi can be performed under liquid conditions.
  • nuc-1 C. elegans strain is more sensitive to RNAi than the wild-type N2 strain. This is most clear for less penetrant phenotypes such as
  • a set of primers for each gene was designed on the basis of sequence data available in the publicly accessible C. elegans sequence database (Acedb) .
  • the cosmid names relate to:
  • PCR was performed on genomic DNA of N2 strain C. elegans to give PCR products of the following si zes :
  • PCR fragments of C04H5.6, KllD9.2b and Y57G11C.15 were linked to BstXI adaptors (Invitrogen) and then cloned into the pGN29 vector cut with SstXI.
  • pGN29 contains two T7 promoters and two T7 terminators flanking a cloning site which is adapted for facilitated cloning of PCR fragments, comprising a stuffer DNA flanked by two BstXI sites (see schematic Figure 3) .
  • the resulting plasmids were designated pGX22 (C04H5.6), pGX52 (KllD9.2b) and pGX104 (Y57G11C.15) .
  • telomere sequence was cloned into pGN39 via recombination sites based on the GATEWAYTM cloning system (Life Technologies, Inc) .
  • pGN39 contains two T7 promoters and two T7 terminators flanking a cloning site which facilitates "High Throughput" cloning based on homologous recombination rather than restriction enzyme digestion and ligation.
  • the cloning site comprises afctRl and a tR2 recombination sites from bacteriophage lambda flanking a gene which is lethal to E. coli, in this case the ccdB gene.
  • This cloning site is derived from the GatewayTM cloning system commercially available from Life Technologies, Inc.
  • the GatewayTM cloning system has been extensively described by Hartley et al . in WO 96/40724 (PCT/US96/10082) .
  • Example 5
  • the screen was performed in unc-31 ⁇ e928) mutant background, to ensure high amounts of dye in the gut lumen, since unc-31 mutations show constitutive pharyngeal pumping.
  • the dye (BCECF-AM: 2 ',7' bis (2- carboxyethyl) -5- (and-6) -carboxyfluorescein, acetoxymethylester) , obtained from Molecular Probes, is cleaved by intracellular esterases. Fluorescence accumulates in the gut cells upon passage through the apical gut membrane.
  • BCECF-AM Mutations with increased staining of the gut cells after 15-30 minutes exposure to the dye were selected and singled on small agar plates.
  • nuc-1 x gun/+ nuc-1/0 or +/+; nuc-1/0 males
  • SEQ ID NO: 1 complete sequence of pGNl
  • SEQ ID NO: 2 complete sequence of pGN8
  • SEQ ID NO: 3 complete sequence of pGN29
  • SEQ ID NO: 4 complete sequence of pGN39
  • SEQ ID NO: 5 complete sequence of pGX22
  • SEQ ID NO: 6 complete sequence of pGX52
  • SEQ ID NO: 7 complete sequence of pGX104
  • SEQ ID NO: 8 complete sequence of pGZ8

Abstract

L'invention concerne des procédés destinés à améliorer l'inhibition d'ARN à double brin par inhibition de l'expression génétique dans des vers nématodes tels que C. elegans. L'invention concerne en particulier la découverte énonçant que des modifications du contexte génétique de C. elegans entraînent une sensibilité accrue à l'inhibition d'ARN à double brin.
PCT/EP2000/013149 1999-12-24 2000-12-22 Inhibition d'arn a double brin WO2001048183A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU21715/01A AU2171501A (en) 1999-12-24 2000-12-22 Improvements relating to double-stranded rna inhibition

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9930691.2A GB9930691D0 (en) 1999-12-24 1999-12-24 Improvements relating to double-stranded RNA inhibition
GB9930691.2 1999-12-24

Publications (2)

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