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 an embryonic zebrafish cell in vivo comprising supplying the cell with a double stranded RNA 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., and provided that, when the double stranded RNA is supplied to the cell by delivery to the cell of double stranded RNA, the double stranded RNA is formed from single-stranded RNA that is purified in the absence of phenol or chloroform.
2. The method of claim 1 , wherein the target gene is an endogenous gene.
3. The method of claim 1 , wherein the target gene is a foreign gene.
4. The method of claim 1 , wherein the target gene is a chromosomal gene.
5. The method of claim 1 , wherein the target gene is an extrachromosomal gene.
6. The method of claim 1 , wherein the target gene is from a pathogen capable of infecting the embryonic zebrafish cell.
7. The method of claim 6 , wherein the pathogen is selected from the group consisting of a virus, bacterium, fungus or protozoan.
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 comprises a nucleotide sequence that is complementary to a region of at least about 25 bases of the target gene.
10. The method of claim 1 , wherein the double stranded RNA is supplied in an amount sufficient to completely inhibit expression of the target gene.
11. The method of claim 1 in which the double stranded RNA comprises a single strand comprising self-complementary portions.
12. The method of claim 1 in which the double stranded RNA comprises two separate complementary strands.
13. The method of claim 1 , wherein the embryonic zebrafish cell is supplied with the double stranded RNA using microinjection.
14. The method of claim 1 , wherein supplying the double stranded RNA to the embryonic zebrafish cell comprises delivering double-stranded RNA to the embryonic zebrafish cell, and wherein the double stranded RNA is treated with RNAse prior to delivery to the embryonic zebrafish cell.
15. The method of claim 1 , wherein supplying the double stranded RNA to the embryonic zebrafish cell comprises delivering double stranded RNA to the embryonic zebrafish cell, the method further comprising, prior to delivering the double stranded RNA to the embryonic zebrafish cell, annealing two complementary single stranded RNAs to yield the double stranded RNA.
16. The method of claim 15 , wherein the single stranded RNAs are annealed in the presence of potassium chloride.
17. The method of claim 1 , wherein the function of the target gene is unknown.
18. The method of claim 1 further comprising introducing into the embryonic zebrafish cell a second double stranded RNA in an amount sufficient to 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.
19. The method of claim 1 comprising introducing into the embryonic zebrafish cell multiple double stranded RNAs in an amount sufficient to 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.
20. The method of claim 1 further comprising identifying a phenotypic change in the zebrafish associated with attenuated expression of the target gene.
21. A method for attenuating the expression of a target gene in an embryonic zebrafish cell comprising:
annealing two complementary single stranded RNAs in the presence of potassium chloride to yield double stranded RNA;
contacting the double stranded RNA with RNAse to purify the double stranded RNA by removing single stranded RNA; and
introducing the purified double stranded RNA into the cell 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., and wherein the double stranded RNA is formed from single-stranded RNA that is purified in the absence of phenol or chloroform.
22. 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.
23. A method for attenuating the expression of a target gene in an embryonic zebrafish cell in vivo comprising delivering a double stranded RNA to the embryonic zebrafish cell in an amount sufficient to specifically attenuate expression of the target gene, wherein the double stranded RNA comprises a nucleotide sequence that is complementary to a region of at least 25 nucleotides of the target gene, and wherein the double stranded RNA is formed from single-stranded RNA that is purified in the absence of phenol or chloroform.
24. The method of claim 23 , wherein the target gene is associated with a disease.
25. The method of claim 23 , wherein the target gene is associated with a disease from a pathogen.
26. The method of claim 1 , wherein the double stranded RNA is supplied to the embryonic zebrafish cell by delivering to the cell a DNA encoding the double stranded RNA.
27. The method of claim 1 , wherein the double stranded RNA has a length of less than about 200 bases.
28. The method of claim 11 , wherein the double stranded RNA comprises a nucleotide sequence that is complementary to a region of at least about 25 bases of the target gene.
29. The method of claim 12 , wherein the double stranded RNA comprises a nucleotide sequence that is complementary to a region of at least about 25 bases of the target gene.
30. A method for attenuating the expression of a target gene in an embryonic fish cell in vivo comprising supplying the cell with a double stranded RNA 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., and provided that, when the double stranded RNA is supplied to the cell by delivery to the cell of double stranded RNA, the double stranded RNA is formed from single-stranded RNA that is purified in the absence of phenol or chloroform.