SELECTING AND STABILIZING dsRNA CONSTRUCTS

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.

1 - 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.