Stable transformation of a population and a method of biocontainment using haploinsufficiency and underdominance principles

Abstract

The present invention relates to a method for reducing the competitive fitness of an organism hemizygous for a transgenic locus compared to the organism homozygous for the transgenic locus comprising the steps of: (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism; and (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism, yielding an organism which is less competitively fit if hemizygous for the transgenic locus than if homozygous for the transgenic locus. The present invention also relates to a method for decreasing the introgression of a transgenic locus in an organism into a population of otherwise interfertile sexually reproducing organisms comprising the steps of: (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism; (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism, and (c) using a transgenic organism obtained in step (b) in an environment comprising otherwise interfertile sexually reproducing wildtype individuals of the organism, wherein the competitive fitness of hemizygous progeny is reduced, thereby decreasing the rate of sexually reproduction and/or viability and/or the competitive fitness of hemizygous progeny. Further envisaged are corresponding genetic systems and genetically modified organisms.

1 . Method for reducing the competitive fitness of an organism hemizygous for a transgenic locus compared to the organism homozygous for the transgenic locus, with the proviso that the organism is not a human being, comprising the steps of:
 (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism; and   (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism,   yielding an organism which is less competitively fit if hemizygous for the transgenic locus than if homozygous for the transgenic locus.   
     
     
         2 . The method of  claim 1 , additionally comprising (i) the step of releasing a transgenic organism obtained in step (b) into a population of the same species such that the transgenic construct is established at a high frequency at a locus in the population; or (ii) the step of using a transgenic organism obtained in step (b) in an environment comprising otherwise interfertile sexually reproducing wildtype individuals of the organism, whereby the competitive fitness of hemizygous progeny is reduced. 
     
     
         3 . (canceled) 
     
     
         4 . Method for the transformation of a population of sexually reproducing organisms, with the proviso that said organism is not a human being, comprising the steps of:
 (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism;   (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism, and   (c) releasing homozygous organisms obtained in the preceding step into a population of the same species such that the transgenic locus is established at a high frequency in the population.   
     
     
         5 . The method of  claim 2 , wherein said releasing step comprises the release, in a single or over multiple generations, of sufficient relative numbers of organisms to result in a frequency in population of the same species greater than the unstable allelic equilibrium frequency predicted by the competitive fitness. 
     
     
         6 . Method for decreasing the introgression of a transgenic locus in an organism into a population of otherwise interfertile sexually reproducing organisms, with the proviso that said organism is not a human being, comprising the steps of:
 (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism;   (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism; and   (c) using a transgenic organism obtained in step (b) in an environment comprising otherwise interfertile sexually reproducing wildtype individuals of the organism, wherein the competitive fitness of hemizygous progeny is reduced, thereby decreasing the rate of sexually reproduction and/or viability of hemizygous progeny.   
     
     
         7 . Method for reducing the size of wild populations of otherwise interfertile sexually reproducing organisms, with the proviso that said organism is not a human being, comprising the steps of:
 (a) reducing the expression of a haploinsufficient gene in the organism, wherein said reduction is conveyed by a transgenic locus in the organism;   (b) rescuing the reduced expression in the organism, wherein said rescue is conveyed by the same transgenic locus in the organism; and   (c) using a hemizygous transgenic organism obtained in step (b) or a mixture of homozygous and hemizygous transgenic organisms obtained in step (b) in an environment comprising otherwise interfertile sexually reproducing wildtype individuals of the organism, wherein the competitive fitness of hemizygous progeny is reduced, thereby decreasing the rate of sexually reproduction and/or viability of hemizygous progeny.   
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein said reducing the expression of a haploinsufficient gene comprises the provision of a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein said rescuing the expression of a haploinsufficient gene comprises a modification of the haploinsufficient gene sequence. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein said transgenic locus comprises an underdominant construct, wherein said underdominant construct comprises a sequence leading to the provision of a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence. 
     
     
         16 .- 20 . (canceled) 
     
     
         21 . The method of  claim 15 , wherein said underdominant construct additionally comprises a modified version of the haploinsufficient gene, which is resistant to a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence. 
     
     
         22 . The method of  claim 1 , wherein said method comprises the initial transformation of the organism with an independent transgenic construct comprising a modified version of the haploinsufficient gene, which is resistant to a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence, followed by the transformation of the organism with an underdominant construct comprising a sequence leading to the provision of a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence and a haploinsufficient gene that comprises the provision of an at least partial rescue to a means which specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or which specifically disrupts the haploinsufficient gene DNA sequence. 
     
     
         23 . (canceled) 
     
     
         24 . The method of  claim 22 , additionally comprising the steps of obtaining organisms homozygous for said underdominant construct; and removing said independent transgenic construct by chromosomal recombination or segregation. 
     
     
         25 .- 30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein additionally a mechanistically distinct population transformation construct is introduced into the organism. 
     
     
         32 . The method of  claim 15 , wherein said underdominant construct additionally comprises an effector gene. 
     
     
         33 . (canceled) 
     
     
         34 . A genetic system comprising the following components:
 (a) a means for specifically reducing the expression of a haploinsufficient gene; and   (b) a rescuing agent able to increase the reduced expression of said haploinsufficient gene.   
     
     
         35 . The genetic system of  claim 34 , wherein said means for specifically reducing the expression of said haploinsufficient gene specifically degrades or directly inactivates the haploinsufficient gene transcript or expression product, or specifically disrupts the haploinsufficient gene DNA sequence. 
     
     
         36 .- 47 . (canceled) 
     
     
         48 . A genetically modified organism, with the proviso that said organism is not a human being, comprising
 (a) a means for specifically reducing the expression of a haploinsufficient gene; and   (b) a rescuing agent able to increase the reduced expression said haploinsufficient gene.   
     
     
         49 .- 60 . (canceled) 
     
     
         61 . The method of  claim 1 , wherein said haploinsufficient gene is an endogenous cytoplasmic ribosomal protein (CRP), a transcription factor, a tumor suppressor gene, a gene related to muscle function, a homeodomain protein coding gene, or another gene with evidence indicating that it is potentially haploinsufficient in a specific organism, preferably Rpl14 or Rpl23aA. 
     
     
         62 . (canceled) 
     
     
         63 . The method of  claim 1 , wherein said organism is a disease vectoring animal, disease causing animal, or a livestock animal, or a plant, or a fungus, or a protist. 
     
     
         64 .- 68 . (canceled) 
     
     
         69 . The method of  claim 63 , wherein said plant is an agricultural plant such as a staple crop, preferably a grain crop, root crops, tubers, pulses, sorghum, or legumes; or a sugar producing crop, preferably sugar cane or sugar beet; or an oil producing plant, preferably rapeseed, soybean, oil palm, safflower, or sunflower; or a pest plant such as an invasive plant, a poison plant or a plant causing allergenic reactions in animals, preferably in human beings. 
     
     
         70 .- 75 . (canceled)