Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA
View Patent ↗Introduction of double stranded RNA into cells, cell culture, organs and tissues, and whole organisms, particularly vertebrates, specifically attenuates gene expression.
1. A method for attenuating the expression of a target gene in a cell, the method comprising contacting an embryonic zebrafish, embryonic chick or fibroblast murine cell with a double stranded RNA (dsRNA) in an amount sufficient to specifically attenuate expression of the target gene, wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.
2. The method of claim 1 , wherein said dsRNA has a length of less than about 200 bases.
3. The method of claim 1 , wherein said dsRNA has a length of at least about 25 bases.
4. The method of claim 1 , wherein the target gene is an endogenous gene.
5. The method of claim 1 , wherein the target gene is a foreign gene.
6. The method of claim 1 , wherein the target gene is a chromosomal gene.
7. The method of claim 1 , wherein the target gene is an extrachromosomal gene.
8. The method of claim 1 , wherein the double stranded RNA comprises a nucleotide sequence that is complementary to the nucleotide sequence of at least a portion of the target gene.
9. The method of claim 1 , wherein the double stranded RNA is supplied in an amount sufficient to completely inhibit expression of the target gene.
10. The method of claim 1 , wherein the double stranded RNA comprises a single strand comprising self-complementary portions.
11. The method of claim 1 , wherein the double stranded RNA comprises two separate complementary strands.
12. The method of claim 1 , wherein the double stranded RNA is supplied to the cell by delivery to the cell of the double stranded RNA.
13. The method of claim 12 , wherein the double stranded RNA is purified in the absence of phenol or chloroform.
14. The method of claim 1 , wherein the double stranded RNA is supplied to the cell by delivering to the cell a DNA encoding the double stranded RNA.
15. The method of claim 1 , wherein the double stranded RNA is treated with RNAse prior to delivery to the cell.
16. The method of claim 1 , wherein supplying the double stranded RNA to the cell comprises delivering double stranded RNA to the cell, the method further comprising, prior to delivering the double stranded RNA to the cell, annealing two complementary single stranded RNAs to yield the double stranded RNA.
17. The method of claim 1 , wherein the single stranded RNAs are annealed in the presence of potassium chloride.
18. The method of claim 1 , wherein the function of the target gene is unknown.
19. The method of claim 1 further comprising supplying the cell with a second double stranded RNA in an amount sufficient to specifically attenuate expression of a second target gene, wherein one of the strands of the second double stranded RNA is capable of hybridizing to the second target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.
20. The method of claim 1 further comprising supplying the cell with multiple double stranded RNAs in an amount sufficient to specifically attenuate expression of multiple target genes, wherein one strand of each double stranded RNA is capable of hybridizing to the corresponding target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.
21. The method of claim 1 further comprising identifying a phenotypic change in the cell associated with attenuated expression of the target gene.
22. The method of claim 1 , wherein the target gene is associated with a disease.
23. The method of claim 1 , wherein the murine cell is a rat cell.
24. The method of claim 1 , wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 70° C.
25. The method of claim 1 , wherein said cell is an embryonic zebrafish cell.
26. The method of claim 1 , wherein said cell is an embryonic chick cell.
27. The method of claim 1 , wherein said cell is a fibroblast murine cell.