Rna interference using a universal target

Abstract

The present invention provides novel methods for manipulating levels of expression of gene products using RNA interference (RNAi). The methods disclosed can be used to investigate gene function, to create disease-resistant organisms, and to treat disease.

1 . A method of reducing the endogenous expression of a plurality of gene products in cells or organisms that exhibit transitive RNA interference comprising:
 providing a plurality of expression cassettes that direct the expression of recombinant transcripts, each transcript having a unique nucleotide sequence encoding an individual gene product and a universal RNA interference target sequence that is 3′ of, and adjacent to, said unique nucleotide sequence encoding an individual gene product,   creating a plurality of transfected test cells by introducing said expression cassettes into cells capable of transcribing and translating the encoded individual gene products, and   introducing an RNA into said transfected test cells, said RNA being capable of inducing RNA interference by specifically targeting the universal RNA interference target sequence shared by the recombinant transcripts;   whereupon the expression of all recombinantly expressed gene products is reduced by a primary RNA interference response, and the expression of homologous endogenously expressed gene products is reduced by a transitive RNA interference response.   
   
   
       2 . The method of  claim 1 , wherein the step of introducing said RNA is by way of introducing a DNA that directs the in vivo transcription of said RNA. 
   
   
       3 . The method of  claim 2 , wherein the RNA transcribed in vivo is a small hairpin RNA. 
   
   
       4 . The method of  claim 1 , wherein the step of introducing said RNA is by way of introducing an RNA synthesized in vitro. 
   
   
       5 . The method of  claim 4 , wherein the RNA synthesized in vitro is a small single-stranded hairpin RNA. 
   
   
       6 . The method of  claim 4 , wherein the RNA synthesized in vitro is a double-stranded siRNA. 
   
   
       7 . The method of  claim 1 , wherein said universal RNA interference target sequence is located in the 3′ untranslated region of the recombinant transcript. 
   
   
       8 . The method of  claim 1 , wherein said universal RNA interference target sequence encodes a peptide, is cloned in frame with the 3′ end of said nucleotide sequence encoding an individual gene product, and results in the expression of a fusion protein. 
   
   
       9 . A method of treating a disease in plants caused by an infectious agent comprising the steps of:
 administering to a plant in need of such treatment an expression cassette comprising a nucleotide sequence encoding a gene product of said infectious agent and a universal RNA interference target sequence, and   introducing into the plant an RNA that induces RNA interference of expression of said gene product of said infectious agent by targeting said universal RNA interference target sequence.   
   
   
       10 . The method of  claim 9 , wherein the step of introducing said RNA that induces RNA interference is by way of introducing a DNA that directs the in vivo transcription of the RNA. 
   
   
       11 . The method of  claim 10 , wherein the RNA transcribed in vivo is a small hairpin RNA. 
   
   
       12 . The method of  claim 9 , wherein the step of introducing said RNA that induces RNA interference is by way of introducing an RNA synthesized in vitro. 
   
   
       13 . The method of  claim 12 , wherein the RNA synthesized in vitro is a small single-stranded hairpin RNA. 
   
   
       14 . The method of  claim 12 , wherein the RNA synthesized in vitro is a double-stranded siRNA. 
   
   
       15 . The method of  claim 12 , wherein the RNA synthesized in vitro is enzymatically synthesized. 
   
   
       16 . The method of  claim 12 , wherein the RNA synthesized in vitro is chemically synthesized. 
   
   
       17 . The method of  claim 9 , wherein said disease is a viral infection and said infectious agent is the virus that causes said viral infection. 
   
   
       18 . The method of  claim 9 , wherein said disease is a fungal infection and said infectious agent is the fungus that causes said fungal infection. 
   
   
       19 . The method of  claim 9 , wherein said disease is caused by nematodes and said infectious agent is the nematode that causes the disease. 
   
   
       20 . A method of imparting on plants a resistance to a pathogen comprising the steps of:
 administering to a plant in need of resistance to a pathogen an expression cassette that directs the expression of a chimeric RNA comprising a nucleotide sequence encoding a gene product of said pathogen, or a portion thereof, operably linked to a universal RNA interference target sequence, and   introducing into the plant an RNA that induces RNA interference of expression of said gene product of said pathogen by targeting said universal RNA interference target sequence.