Gene Expression System

Abstract

Two or more conditional, dominant, lethal gene expression systems provide high levels of penetrance in insects. Lethality is induced at an earlier stage of development and the risk of biochemical resistance is reduced, as compared to a single insect conditional, dominant, lethal gene expression system. The invention is useful for the control of insect populations.

1 - 32 . (canceled) 
     
     
         33 . A polynucleotide comprising a first and a second gene expression system, wherein:
 i) the first gene expression system comprises the following components: a first dominant lethal gene operably linked to a first promoter, a gene encoding a first activating transcription factor, and a first splice control sequence, and   ii) the second gene expression system comprises the following components: a second dominant lethal gene operably linked to a second promoter, a gene encoding a second activating transcription factor, and a second splice control sequence,   wherein:   each of said activating transcription factors is capable of activating at least one of said promoters, provided that both of said promoters are activated when both of said transcription factors are expressed,   each of the first and second splice control sequences mediates female-specific expression of the first and second dominant lethal genes, respectively, by alternative splicing,   the transactivation activity of each of the first and second activating transcription factors is repressible by a first and a second exogenous control factor, respectively, wherein said first exogenous control factor is the same as or different from said second exogenous control factor, and   each of said components of said first gene expression system are the same as or different from said components of said second gene expression system.   
     
     
         34 . The polynucleotide according to  claim 33 , wherein the first activating transcription factor is the gene product of the first dominant lethal gene and/or the second activating transcription factor is the gene product of the second dominant lethal gene, such that said transcription factor also provides the lethal effect conferred by said dominant lethal gene. 
     
     
         35 . The polynucleotide according to  claim 33 , wherein one or both of the first and second splice control sequences mediates female-specific expression of the respective dominant lethal gene by, together with a spliceosome, mediating splicing of a RNA transcript of the respective dominant lethal gene to produce a mRNA coding for a functional protein and at least one mRNA coding for a non-functional protein, wherein the mRNA coding for a functional protein is produced in a female. 
     
     
         36 . The polynucleotide according to  claim 33 , wherein each of the first and second promoters is substantially inactive in the absence of the activating transcription factor capable of activating said promoter. 
     
     
         37 . The polynucleotide according to  claim 33 , wherein one or both of the first and second lethal genes is, independently, tTA or a tTAV gene variant. 
     
     
         38 . The polynucleotide according to  claim 33 , wherein one or both of the first and second splice control sequences is, independently, derived from a tra intron, dsx gene, or Actin-4 gene. 
     
     
         39 . The polynucleotide according to  claim 33 , wherein one or both of the first and second gene expression systems further comprises an enhancer. 
     
     
         40 . The polynucleotide according to  claim 33 , wherein one or both of the first and second promoters is expressed during at least embryonic development. 
     
     
         41 . The polynucleotide according to  claim 33 , wherein one of the first and second gene expression systems comprises:
 (a) a third dominant lethal gene; and   (b) a third promoter, wherein the third promoter is operably linked to the third dominant lethal gene, wherein the activating transcription factor capable of activating the promoter of said gene expression system is also capable of activating the third promoter.   
     
     
         42 . The polynucleotide according to  claim 41 , wherein one of the first and second gene expression systems further comprises an enhancer associated with the promoter of said gene expression system, wherein the third promoter is also associated with said enhancer. 
     
     
         43 . The polynucleotide according to  claim 33 , wherein the polynucleotide further comprises a fluorescent marker. 
     
     
         44 . The polynucleotide according to  claim 33 , wherein:
 the first dominant lethal gene is tTAV (SEQ ID NO: 26), the first activating transcription factor is the tTAV gene product, the first promoter is Hsp70, the first splice control sequence is Cctra,   the second dominant lethal gene is tTAV3 (SEQ ID NO: 28), the second activating transcription factor is the tTAV3 gene product, the second promoter is srya and the second splice control sequence is Bztra,   the first gene expression system further comprises a first enhancer associated with the first promoter, wherein the first enhancer is tetOx7,   the second gene expression system further comprises a second enhancer associated with the second promoter, wherein the second enhancer is tetOx14, the polynucleotide further comprises a third dominant lethal gene operably linked to a third promoter, the third promoter being associated with the second enhancer, wherein the third dominant lethal gene is VP16 and the third promoter is Hsp70, wherein the second promoter is associated with one end of the second enhancer and the third promoter is associated with the other end of the second enhancer,   the polynucleotide further comprises a first genetic marker, which is DsRed2.   
     
     
         45 . A genetic construct for transforming an insect, comprising the polynucleotide as described in  claim 33 . 
     
     
         46 . The genetic construct according to  claim 45 , wherein the construct further comprises at least four transposon inverted repeats, forming at least two pairs of opposing transposon inverted repeats, wherein said polynucleotide is located between two pairs of opposing transposon inverted repeats such that excision by a transposase or transposases of said pairs, in situ, is effective to be able to leave said polynucleotide integrated in the host genome, without flanking transposon inverted repeats in the host genome. 
     
     
         47 . The genetic construct according to  claim 46 , wherein each of the transposon inverted repeats bounding said polynucleotide is a minimal terminal inverted repeat. 
     
     
         48 . The genetic construct according to  claim 46 , wherein four of the at least four transposon inverted repeats form a first and a second pair of opposing inverted repeats, wherein:
 the four transposon inverted repeats are piggyBac inverted repeats,   the first pair consists of an internal 3′ piggyBac end proximal the polynucleotide and an external 5′ piggyBac end distal the polynucleotide, and   the second pair consists of an internal 5′ piggyBac end proximal the polynucleotide and an external 3′ piggyBac end distal the polynucleotide.   
     
     
         49 . The genetic construct according to  claim 48 , wherein:
 the internal 3′ piggyBac end has a nucleic acid sequence consisting of SEQ ID NO: 32;   the external 5′ piggyBac end has a nucleic acid sequence consisting of SEQ ID NO:30;   the internal 5′ piggyBac end has a nucleic acid sequence consisting of SEQ ID NO: 30; and   the external 3′ piggyBac end has a nucleic acid sequence consisting of SEQ ID NO: 31.   
     
     
         51 . The genetic construct according to  claim 46 , further comprising at least one genetic marker between the transposon inverted repeats of at least one pair of opposing transposon inverted repeats. 
     
     
         51 . An insect comprising the polynucleotide as described in  claim 33 , wherein the insect is a Tephritidae, Drosophilidae, Lonchaeidae, Pallopteridae, Platystomatidae, Pyrgotidae, Richardiidae, or Ulidiidae. 
     
     
         52 . The insect according to  claim 51 , wherein the insect is  Ceratitis capitata.    
     
     
         53 . A method of controlling an insect population comprising release of the insect as described in  claim 51 .