SELECTING AND STABILIZING dsRNA CONSTRUCTS
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Abstract
The invention provides methods for selecting nucleotide sequences that yield dsRNA-mediated gene suppression in a target organism and enable their uptake by the target organism. The invention further provides expression constructs that confer stabilized expression of such sequences in a transgenic host cell, and methods for their use. Also provided are organisms, cells and tissues prepared by a method of the invention.
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Citations
96 Claims
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1-40. -40. (canceled)
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41. A method of increasing the pest or pathogen-inhibitory activity of a dsRNA, comprising:
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a) obtaining a first nucleic acid segment that when expressed as a dsRNA and taken up by a target crop pest or pathogen inhibits feeding by the target crop pest or pathogen or progeny thereof; and b) linking the first nucleic acid segment to a second nucleic acid segment to create a longer nucleic acid segment, wherein the second nucleic acid segment is a nucleic acid that does not inhibit feeding by the target crop pest or pathogen or progeny thereof when expressed as a dsRNA, and wherein a dsRNA expressed from the longer nucleic acid exhibits increased potency of inhibition of feeding by the target crop pest or pathogen or progeny thereof relative to the dsRNA expressed from the first nucleic acid segment alone. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63)
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50-51. -51. (canceled)
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53-54. -54. (canceled)
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64. A method of producing an expression construct for expressing a dsRNA with increased specificity of pest or pathogen-inhibitory activity comprising:
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a) obtaining a starting nucleic acid molecule substantially complementary to at least a first coding sequence of a target crop pest or pathogen; b) selecting a region within the starting nucleic acid molecule that when expressed as a dsRNA inhibits feeding by the target crop pest or pathogen or progeny thereof following uptake of the dsRNA expressed from the region by the target crop pest or pathogen; c) linking the region to a second nucleic acid molecule to produce an expression construct, wherein the second nucleic acid molecule when expressed as a dsRNA does not inhibit feeding by a target crop pest or pathogen or progeny thereof following uptake of the dsRNA. - View Dependent Claims (65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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- 83. A method of enhancing the control of a target crop pest or pathogen in a plant comprising expressing in the cells of the plant at least two dsRNA sequences that function upon uptake by the pest or pathogen to inhibit the expression of at least a first target coding sequence within the target crop pest or pathogen, wherein the two dsRNA sequences are substantially complementary to two non-contiguous portions of the first target coding sequence or to two different coding sequences of the target crop pest or pathogen.
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85-86. -86. (canceled)
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96-98. -98. (canceled)
Specification