SELECTING AND STABILIZING dsRNA CONSTRUCTS

• US 20140080755A1
• Filed: 08/06/2013
• Published: 03/20/2014
• Est. Priority Date: 02/13/2006
• Status: Abandoned Application

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Heck, Gregory R., Munyikwa, Tichafa R.I., Roberts, James K., Johnson, Scott C., Vaughn, Ty T., Goley, Jean C.
• Application Number:
  US13/960,646
• Publication Number:
  US 20140080755A1
• Time in Patent Office: Days
• US Class Current: 514/4.5
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/10 in screening processes
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

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.

Claims

1-95. -95. (canceled)

96. A method for managing crop pest resistance to an agent for controlling the crop pest, comprising contacting the crop pest with a nucleic acid segment produced by a method comprising:

(a) obtaining a starting nucleic acid molecule substantially complementary to a target gene;

(b) preparing a plurality of nucleic acid segments from the starting nucleic acid molecule;

(c) assaying the nucleic acid segments for the ability to suppress expression of the target gene when expressed as a dsRNA in a cell comprising the target gene; and

(d) identifying at least a first nucleic acid segment from the plurality of nucleic acid segments that provides a desired level of suppression of the target gene when expressed as a dsRNA,

and one additional agent selected from the group consisting of a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a biocontrol agent, and an insecticide.

97. The method of claim 96, wherein the insecticide is selected from the group consisting of a carbaryl insecticide, fenvalerate, esfenvalerate, malathion, a carbofuran insecticide, chloropyrifos, fonophos, phorate, terbufos, permethrin, a neonicotinoid, and tefluthrin.

98. The method of claim 96, wherein the additional agent is provided as a seed treatment.

99. The method of claim 96 wherein the additional agent is a Bacillus thuringiensis insecticidal protein.

100. The method of claim 99, wherein the Bacillus thuringiensis insecticidal protein is selected from the group consisting of a Cry1, a Cry3, a TIC851, a CryET70, a Cry2, ET29, ET37, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, a binary insecticidal protein ET29 and TIC810, a binary insecticidal protein ET37 and TIC812, and a binary insecticidal protein PS 149B 1.

101. The method of claim 96, wherein the desired level of suppression is complete suppression of the target gene.

102. The method of claim 96, wherein the desired level of suppression is incomplete suppression of the target gene.

103. The method of claim 96, wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises expressing the segments as a dsRNA in a cell comprising the target gene and determining the level of suppression of the target gene.

104. The method of claim 96, wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises expressing the segments as a dsRNA in a cell;

allowing a pest comprising the target gene to feed on the plant cell; and

determining the level of suppression of the target gene.

105. The method of claim 96, wherein assaying the nucleic segments for the ability to suppress the target gene comprises calculating a Reynolds score for the nucleic acid segments.

106. The method of claim 96, wherein assaying the nucleic acid segments for the ability to suppress the target gene comprises providing said segments as dsRNA molecules in the diet of an organism comprising the target gene and determining the level of suppression of the target gene.

107. The method of claim 106, wherein determining the level of suppression of the target gene comprises observing morbidity, mortality, or stunting of said organism.

108. The method of claim 96, wherein the crop pest is an insect.

109. The method of claim 108, wherein the insect is selected from the group consisting of a Coleopteran, a Lepidopteran, a Hemipteran, and a Homopteran insect.

110. The method of claim 108, wherein the crop pest is a Diabrotica spp.

111. The method of claim 96, wherein target crop pest is a nematode.