Method of regulating gene expression

Abstract

The present invention relates, in general, to gene expression and, in particular, to a method of inhibiting the expression of a target gene and to constructs suitable for use in such a method.

1 .- 30 . (canceled) 
     
     
         31 . A DNA construct comprising a polymerase II-based promoter functional in said cell operably linked to a nucleic acid comprising a nucleotide sequence encoding, as part of a longer encoded sequence, an miRNA precursor, said miRNA precursor comprising a stem loop structure and comprising in said stem of said stem loop structure a sequence complementary to a portion of an RNA transcript of a target gene, wherein said stem of said stem loop structure is about 19-45 base pairs long and the loop of said stem loop structure is about 4-25 nucleotides. 
     
     
         32 . The DNA construct according to  claim 31 , wherein said loop of said stem loop structure of said miRNA precursor comprises a sequence corresponding to a naturally occurring miRNA. 
     
     
         33 . The DNA construct according to  claim 31 , wherein the base of said stem of said stem loop structure of said miRNA precursor comprises a base paired region at least 3 base pairs in length. 
     
     
         34 . The DNA construct according to  claim 31 , wherein said stem of said stem loop structure of said miRNA precursor includes at least one bulge. 
     
     
         35 . The DNA construct of  claim 34 , wherein the bulge is not present within the last three base pairs at the base of said stem. 
     
     
         36 . The DNA construct according to  claim 31 , wherein said construct is present in a viral vector. 
     
     
         37 . The DNA construct according to  claim 31 , wherein said construct is present in a plasmid. 
     
     
         38 . The DNA construct according to  claim 31 , wherein said loop of said stem loop structure of said miRNA precursor comprises 6-15 nucleotides. 
     
     
         39 . The DNA construct according to  claim 31 , wherein the polymerase II-based promoter is a constitutive promoter. 
     
     
         40 . The DNA construct according to  claim 31 , wherein the polymerase II-based promoter is an inducible promoter. 
     
     
         41 . The DNA construct according to  claim 31 , wherein the polymerase II-based promoter is a tissue-specific promoter.