Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA
AssigneeMedical College of Georgia Research Institute, Inc.
InventorsYin-Xiong Li, Michael J. Farrell, Margaret J. Kirby
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Abstract
Introduction of double stranded RNA into cells, cell culture, organs and tissues, and whole organisms, particularly vertebrates, specifically attenuates gene expression.
Claims30
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.
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.
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.
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.
Dependencies are inferred from the claim text.