Gene silencing

Abstract

Provided are methods of screening for the occurrence of gene silencing (e.g. post transcriptional gene silencing) in an organism (e.g. a plant or animal), which method comprises the steps of: (i) obtaining a sample of material from said organism, (ii) producing a nucleic acid extract from said sample, (iii) analysing said extract such as to determine the presence or absence of short RNA molecules which are approximately 25 nucleotides in length (SRMs) in said nucleic extract, (iv) correlating the presence of said SRMs in the extract with the occurrence of gene silencing in said organism. The SRMs are preferably short anti-sense RNA molecules (SARMs). Also provided are associated methods for detecting the silencing of a target gene in an organism. Processes for isolating one or more RNA molecules, such as SARMs, which may be advantageously employed in the method, may include a purification step selected from (i) filtration; (ii) differential precipitation (iii) ion exchange chromatography, followed by separation the purified RNA molecules according to size by electrophoresis through 15% polyacrylamide gel containing 7M urea as a denaturant and TBE (0.5×) as a buffer, and blotting by electrophoresis. Also provided are processes for isolating silencing agents comprising SRMs; methods of selecting target regions of target genes for directed silencing; and methods of silencing target genes in organisms based on these. Silencing in a first organism may be achieved by generating in a second organism, SRMs which are introduced into the first organism such as to silence a target gene therein. Also provided are DNA constructs, host cells, plants and non-human mammals which comprise target genes which have been silenced in accordance with the methods herein.

1 . A method of detecting the silencing of a target gene in an organism, wherein said silencing is initiated by introduction of an exogenous nucleic acid, which method comprises the steps of: (i) obtaining a sample of material from said organism, (ii) producing a nucleic acid extract from said sample, (iii) analyzing said extract such as to determine the presence or absence of short RNA molecules which are 21-25 nucleotides in length (SRMs) in said extract, (iv) characterizing any SRMs which are present in said extract such as to determine sequence identity or similarity with said target gene, and (v) correlating the presence of said SRMs having sequence identity or similarity with said target gene in the extract with the occurrence of gene silencing in said organism.  
   
   
       2 . A method in accordance with  claim 1 , wherein the organism is a plant.  
   
   
       3 . A method in accordance with  claim 1 , wherein the organism is a nematode.  
   
   
       4 . A method in accordance with  claim 1 , wherein the organism is a mammal.  
   
   
       5 . A method in accordance with  claim 1  wherein the SRMs are short anti-sense RNA molecules (SARMs).  
   
   
       6 . A method in accordance with claim I wherein the SRMs are short sense RNA molecules (SSRMs).  
   
   
       7 . A method in accordance with  claim 1 , wherein the gene silencing is post-transcriptional gene silencing (PTGS).  
   
   
       8 . A method in accordance with  claim 1 , wherein the silencing of said target gene in the organism is associated with pathogen derived resistance.  
   
   
       9 . A method in accordance with  claim 1 , wherein the silencing of said target gene in the organism is associated with modification of a specific trait by co-suppression of the target gene.  
   
   
       10 . A method of identifying a silenced target gene in an organism in which gene silencing is detected as claimed in  claim 1 , which method further comprises the steps of: (vi) preparing a library of genes from said organism, and (vii) identifying those genes in said library which share sequence identity or similarity with any SRMs which are present in the extract as being genes which are silenced in the organism.  
   
   
       11 . A process for isolating one or more RNA molecules associated with target gene silencing from a sample of material from an organism, wherein the RNA molecules are SRMs which share sequence identity with the target gene, and wherein said silencing is initiated by introduction of an exogenous nucleic acid, which process comprises the steps of: (a) producing a nucleic acid extract from said sample, (b) purifying said extract to produce purified RNA molecules which are 21-25 nucleotides in length by carrying out at least one purification step selected from the following steps (i) filtration; (ii) differential precipitation (iii) ion exchange chromatography, such as to isolate said SRMs.  
   
   
       12 . A process according to  claim 11  which further comprises the step of separating the purified RNA molecules according to size by electrophoresis through a gel, which gel is a 15% polyacrylamide gel containing 7M urea as a denaturant and TBE (0.5.times.) as a buffer.  
   
   
       13 . A process according to  claim 12  which further comprises the step of transferring the RNA molecules on the gel to a hybridization membrane by electrophoresis.  
   
   
       14 . A process according to  claim 13  which further comprises the step of labeling RNA molecules on the hybridization membrane using a radioactive probe obtained from a single stranded RNA molecule transcribed in vitro from a plasmid DNA template.  
   
   
       15 . A process for isolating a silencing agent comprising SRMs for a target gene from an organism, which process comprises the steps of: (i) silencing said target gene in said organism, wherein said silencing is initiated by introduction of an exogenous nucleic acid, (ii) obtaining a sample of material from said organism, (iii) performing a process in accordance with  claim 11  to isolate said SRMs.  
   
   
       16 . A method according to  claim 1  wherein the target gene is an animal gene selected from the group consisting of: a gene involved in apoptosis; a gene involved in cell-cycle regulation; and a gene involved in a neurological process.  
   
   
       17 . A method as claimed in  claim 1 , wherein said short RNA molecules are between 23 and 25 nucleotides in length.  
   
   
       18 . A method as claimed in  claim 1 , wherein said short RNA molecules are 25 nucleotides in length.