Characterisation of gene function using double stranded RNA inhibition

Abstract

There is provided a method of identifying DNA responsible for conferring a particular phenotype in a cell which method comprises a) constructing a cDNA or genomic library of the DNA of said cell in a suitable vector in an orientation relative to a promoter(s) capable of initiating transcription of said cDNA or DNA to double stranded (ds) RNA upon binding of an appropriate transcription factor to said promoter(s), b) introducing said library into one or more of said cells comprising said transcription factor, and c) identifying and isolating a particular phenotype of said cell comprising said library and identifying the DNA or cDNA fragment from said library responsible for conferring said phenotype. Using this technique it is also possible to assign function to a known DNA sequence by a) identifying a homologue(s) of said DNA sequence in a cell, b) isolating the relevant DNA homologue(s) or a fragment thereof from said cell, c) cloning said homologue or fragment thereof into an appropriate vector in an orientation relative to a suitable promoter(s) capable of initiating transcription of dsRNA from said DNA homologue or fragment upon binding of an appropriate transcription factor to said promoter(s) and d) introducing said vector into said cell from step a) comprising said transcription factor.

1 . A method for introducing dsRNA into  C. elegans , comprising 
 feeding to  C. elegans  a micro-organism expressing said dsRNA.    
     
     
         2 . A method for introducing a DNA that encodes and is capable of producing dsRNA into  C. elegans , comprising 
 feeding to  C. elegans  a micro-organism comprising said DNA that encodes and is capable of producing dsRNA.    
     
     
         3 . A method according to  claim 2 , in which said DNA is in the form of an expression vector.  
     
     
         4 . A method according to  claim 3 , in which said expression vector comprises a promoter or promoters oriented relative to a DNA sequence such that the promoter or promoters initiate transcription of said DNA sequence to double stranded RNA upon binding of a transcription factor to said promoter or promoters.  
     
     
         5 . A method according to  claim 4 , in which said expression vector comprises two identical promoters flanking said DNA sequence.  
     
     
         6 . A method according to  claim 4 , in which said expression vector comprises said DNA sequence in a sense and an antisense orientation relative to said promoter or promoters.  
     
     
         7 . A method according to  claim 4 , in which said transcription factor is a phage polymerase.  
     
     
         8 . A method according to  claim 7 , in which said promoter(s) is/are selected from the group consisting of T7, T3 and SP6 promoter(s).  
     
     
         9 . A method according to  claim 4 , in which said micro-organism is adapted to express said transcription factor.  
     
     
         10 . A method according to  claim 9 , in which said transcription factor is T7 polymerase.  
     
     
         11 . A method according to  claim 4 , in which said  C. elegans  is adapted to express said transcription factor.  
     
     
         12 . A method according to  claim 11 , in which said transcription factor is T7 polymerase.  
     
     
         13 . A method for down-regulating the expression of a gene of interest in  C. elegans , comprising 
 feeding  C. elegans  with a micro-organism that expresses dsRNA corresponding to the gene of interest.    
     
     
         14 . A method for down-regulating the expression of a gene of interest in  C. elegans , comprising 
 feeding  C. elegans  with a micro-organism that comprises DNA that encodes and is capable of expressing dsRNA corresponding to the gene of interest in  C. elegans.      
     
     
         15 . A method according to  claim 14 , in which said DNA that encodes and is capable of expressing dsRNA corresponding to the gene of interest is in the form of an expression vector that comprises a DNA sequence corresponding to the gene of interest.  
     
     
         16 . A method according to  claim 15 , in which said expression vector comprises a promoter or promoters oriented relative to said DNA sequence such that the promoter or promoters initiate transcription of said DNA sequence to double stranded RNA upon binding of a transcription factor to said promoter or promoters.  
     
     
         17 . A method according to  claim 16 , in which said expression vector comprises two identical promoters flanking said DNA sequence.  
     
     
         18 . A method according to  claim 16 , in which said expression vector comprises said DNA sequence in a sense and an antisense orientation relative to said promoter or promoters.  
     
     
         19 . A method according to  claim 16 , in which said transcription factor is a phage polymerase.  
     
     
         20 . A method according to  claim 19 , in which said promoter(s) is/are selected from the group consisting of T7, T3 and SP6 promoter(s).  
     
     
         21 . A method according to  claim 16 , in which said micro-organism is adapted to express said transcription factor.  
     
     
         22 . A method according to  claim 21 , in which said transcription factor is T7 polymerase.  
     
     
         23 . A method according to  claim 16 , in which said  C. elegans  is adapted to express said transcription factor.  
     
     
         24 . A method according to  claim 23 , in which said transcription factor is T7 polymerase.  
     
     
         25 . A method according to any of claims  1 ,  2 ,  13  or  14 , in which the micro-organism is a bacterium.  
     
     
         26 . A method according to  claim 25 , in which the bacterium is  E. coli.    
     
     
         27 . A method according to  claim 26 , in which the  E. coli  is a RNAse III negative strain.  
     
     
         28 . A method according to any of claims  1 ,  2 ,  13  or  14 , in which the  C. elegans  is DNAse deficient.  
     
     
         29 . A method according to  claim 28 , in which the DNAse deficient  C. elegans  is a nuc-1 mutant.  
     
     
         30 . A micro-organism, comprising an expression vector that encodes and is capable of producing dsRNA, in which said expression vector comprises a promoter or promoters oriented relative to a DNA sequence such that the promoter or promoters initiate transcription of said DNA sequence to double stranded RNA upon binding of a transcription factor to said promoter or promoters.  
     
     
         31 . A micro-organism according to  claim 30 , in which said expression vector comprises two identical promoters flanking said DNA sequence.  
     
     
         32 . A micro-organism according to  claim 30 , in which said expression vector comprises said DNA sequence in a sense and an antisense orientation relative to said promoter or promoters.  
     
     
         33 . A micro-organism according to  claim 30 , in which said transcription factor is a phage polymerase.  
     
     
         34 . A micro-organism according to  claim 33 , in which said promoter(s) is/are selected from the group consisting of T7, T3 and SP6 promoter(s).  
     
     
         35 . A micro-organism according to  claim 30 , wherein said micro-organism is adapted to express said transcription factor.  
     
     
         36 . A micro-organism according to  claim 35 , wherein said transcription factor is T7 polymerase.  
     
     
         37 . A micro-organism according to  claim 30 , in which said DNA sequence has been derived from  C. elegans.    
     
     
         38 . A micro-organism according to  claim 37 , in which said DNA sequence is a  C. elegans -derived cDNA or cDNA fragment.  
     
     
         39 . A micro-organism according to  claim 30 , wherein the micro-organism is a bacterium.  
     
     
         40 . A micro-organism according to  claim 39 , wherein said bacterium is  E. coli.    
     
     
         41 . A micro-organism according to  claim 40 , wherein said  E.coli _is a RNAse III negative strain.  
     
     
         42 . A library of  C. elegans  cDNA or  C. elegans  cDNA fragments, said library having been transformed into a micro-organism by a method comprising: 
 (a) inserting cDNA(s) or cDNA fragment(s) from said  C. elegans  cDNA library into a cloning site of an expression vector, in which said expression vector comprises a promoter or promoters oriented relative to said cloning site such that the promoter or promoters initiate transcription of said cDNA or cDNA fragment inserted in said cloning site to double stranded RNA upon binding of a transcription factor to said promoter or promoters;    (b) transforming the micro-organism with the expression vectors having the cDNA(s) or cDNA fragment(s) inserted therein.    
     
     
         43 . A library according to  claim 42 , in which said expression vector comprises two identical promoters flanking said cDNA or cDNA fragment.  
     
     
         44 . A library according to  claim 42 , in which said expression vector comprises said cDNA or cDNA fragment in a sense and an antisense orientation relative to said promoter or promoters.  
     
     
         45 . A library to according to  claim 42 , in which said transcription factor is a phage polymerase.  
     
     
         46 . A library according to  claim 45 , in which said promoter(s) is/are selected from the group consisting of T7, T3 and SP6 promoter(s).  
     
     
         47 . A library according to  claim 42 , in which said micro-organism is adapted to express said transcription factor.  
     
     
         48 . A library according to  claim 47 , in which said transcription factor is T7 polymerase.  
     
     
         49 . A library according to  claim 42 , in which the micro-organism is a bacterium.  
     
     
         50 . A library according to  claim 49 , in which the bacterium is  E.coli.    
     
     
         51 . A library according to  claim 50 , in which the  E. coli  is a RNAse III negative strain.  
     
     
         52 . A library according to  claim 42 , which is organised into hierarchical pools.  
     
     
         53 . A method for expressing double stranded RNA in  C. elegans , comprising feeding  C. elegans  a library according to any of claims  42 ,  50  and  51 .