Methods and compositions for RNA interference using recombinant Dicer and Argonaut
The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). 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 method for attenuating expression of a target gene in a non-embryonic mammalian cell in culture, comprising introducing into the cell by transfection a double stranded RNA (dsRNA) in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA is about 22 nucleotides in length and complementary across its length to a nucleotide sequences of the target gene and does not activate protein kinase RNA-activated (PKR) sequence-independent response, and wherein the cell is engineered with (i) a recombinant gene encoding a Dicer activity, (ii) a recombinant gene encoding an Argonaut activity, or (iii) both.
2. The method of claim 1 , wherein the recombinant gene encodes a protein which includes an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4 or 5.
3. The method of claim 1 , wherein the recombinant gene includes a coding sequence that hybridizes to SEQ ID NO: 1 or 3 under a wash condition of 2×SSC at 22° C.
4. The method of claim 1 , wherein the target gene is an endogenous gene of the cell.
5. The method of claim 1 , wherein the target gene is an heterologous gene relative to the genome of the cell.
6. The method of claim 1 , wherein expression of the target gene is attenuated by at least 10 fold.
7. The method of claim 1 , wherein the transfection is mediated by calcium precipitating agents.
8. The method of claim 1 , wherein the transfection is mediated by lipid carrier agents.
9. The method of claim 1 , wherein the dsRNA is produced by a vector.
10. The method of claim 5 , wherein the target gene is a pathogen gene.
11. The method of claim 1 , wherein the cell is a primate cell.
12. The method of claim 1 , wherein the cell is a human cell.