Methods and compositions for RNA interference
The present invention provides methods for attenuating gene expression in a cell, especially in a mammalian cell, using gene-targeted double stranded RNA (dsRNA), such as a hairpin RNA. The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
1. A transgenic mouse comprising a germline or somatic cell, wherein the germline or somatic cell comprises a transgene, wherein the transgene comprises a sequence encoding a short hairpin RNA (shRNA) molecule,
wherein the shRNA molecule comprises a double-stranded region, wherein the double-stranded region consists of at least 20 nucleotides but not more than 29 nucleotides,
wherein the shRNA molecule is a substrate for Dicer-dependent cleavage and does not trigger a protein kinase RNA-activated (PK) response in the mammalian cell,
wherein the double-stranded region of the shRNA molecule comprises a sequence that is complementary to a portion of a target gene, and
wherein the shRNA molecule is stably expressed in said germline or somatic cell without use of a PK inhibitor and in an amount sufficient to attenuate expression of the target gene in a sequence specific manner.
2. The transgenic mouse of claim 1 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 21 nucleotides.
3. The transgenic mouse of claim 1 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 22 nucleotides.
4. The transgenic mouse of claim 1 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 25 nucleotides.
5. The transgenic mouse of claim 1 , wherein the short hairpin RNA molecule comprises a double-stranded region consisting of 29 nucleotides.
6. A method for attenuating expression of a target gene in germline or somatic cells of a transgenic mouse, the method comprising:
(a) introducing into a mouse embryonic stem cell a sequence encoding a short hairpin RNA (shRNA) molecule comprising a double-stranded region, wherein the double-stranded region consists of at least 20 nucleotides but not more than 29 nucleotides,
wherein the shRNA molecule is a substrate for Dicer-dependent cleavage,
wherein the double-stranded region of the shRNA molecule comprises a sequence that is complementary to a portion of said target gene,
(b) generating a transgenic mouse from the mouse embryonic stem cell of step (a), said transgenic mouse comprising germline or somatic cells,
wherein the germline or somatic cells comprise a transgene,
wherein the transgene comprises a sequence encoding a short hairpin RNA (shRNA) molecule,
wherein the shRNA molecule is stably expressed in said germline or somatic cells without use of a PK inhibitor and in an amount sufficient to attenuate expression of the target gene in a sequence specific manner,
wherein the shRNA molecule does not trigger a protein kinase RNA-activated (PK) response in the germline or somatic cell,
whereby expression of the target gene is inhibited in said germline or somatic cells.
7. The method of claim 6 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 21 nucleotides.
8. The method of claim 6 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 22 nucleotides.
9. The method of claim 6 , wherein the shRNA molecule comprises a double-stranded region consisting of at least 25 nucleotides.
10. The method of claim 6 , wherein the shRNA molecule comprises a double-stranded region consisting of 29 nucleotides.