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 composition comprising a DNA construct comprising a constitutive or inducible promoter operably linked to a heterologous 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 comprises about 4-25 nucleotides. 
     
     
         32 . The composition of  claim 31 , wherein said construct is such that, upon introduction of said construct into a cell comprising said target gene, said miRNA precursor is transcribed as part of a transcript of said longer encoded sequence which is processed so that said miRNA precursor is excised therefrom, said miRNA precursor is then processed so that a mature miRNA about 21 or 22 nucleotides in length is excised from said miRNA precursor, and inhibition of expression of said target gene is effected. 
     
     
         33 . The composition of  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 composition of  claim 31 , wherein said stem of said stem loop structure of said miRNA precursor includes at least one bulge. 
     
     
         35 . The composition of  claim 31 , wherein said construct is present in a viral vector or a plasmid. 
     
     
         36 . The composition of  claim 31 , wherein said loop of said stem loop structure of said miRNA precursor comprises 6-15 nucleotides. 
     
     
         37 . A DNA construct comprising a polymerase II-based promoter functional in a cell operably linked to a nucleic acid comprising a nucleotide sequence encoding 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. 
     
     
         38 . The DNA construct of  claim 37 , wherein the nucleic acid further comprises a longer encoded sequence with the miRNA precursor encoded within the longer encoded sequence. 
     
     
         39 . The DNA construct according to  claim 37 , wherein said loop of said stem loop structure of said miRNA precursor comprises a sequence corresponding to a naturally occurring miRNA. 
     
     
         40 . The DNA construct according to  claim 37 , 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. 
     
     
         41 . The DNA construct according to  claim 37 , wherein said stem of said stem loop structure of said miRNA precursor includes at least one bulge. 
     
     
         42 . The DNA construct according to  claim 37 , wherein said construct is present in a viral vector or a plasmid. 
     
     
         43 . The DNA construct according to  claim 37 , wherein said loop of said stem loop structure of said miRNA precursor comprises 6-15 nucleotides. 
     
     
         44 . The DNA construct according to  claim 37 , wherein the DNA construct is within a plant cell. 
     
     
         45 . The DNA construct according to  claim 37 , wherein the DNA construct is within an animal cell.