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 . A polynucleotide sequence comprising a first and a second gene expression system, wherein:
 i) the first gene expression system comprises the 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,   ii) the second gene expression system comprises the 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.   
     
     
         2 . A polynucleotide sequence according to  claim 1 , 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. 
     
     
         3 . A polynucleotide sequence according to  claim 1  or  2 , 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. 
     
     
         4 . A polynucleotide sequence according to any one of the preceding claims, wherein each of the first and second promoters is substantially inactive in the absence of the activating transcription factor capable of activating said promoter. 
     
     
         5 . A polynucleotide sequence according to any one of the preceding claims, wherein one or both of the first and second lethal genes is, independently, tTA or a tTAV gene variant, preferably a tTAV gene variant. 
     
     
         6 . A polynucleotide sequence according to  claim 5 , wherein one or both of the first and second dominant lethal genes is, independently, one of tTAV (SEQ ID NO: 26), tTAV2 (SEQ ID NO: 27) and tTAV3 (SEQ ID NO: 28). 
     
     
         7 . A polynucleotide sequence according to any one of the preceding claims, wherein one or both of the first and second splice control sequences is, independently, derived from a tra intron, a dsx gene or an Actin-4 gene, preferably a tra intron. 
     
     
         8 . A polynucleotide sequence according to  claim 7 , wherein one or both of the first and second splice control sequences is derived from a Tephritid, preferably from one of the  Ceratitis, Bactrocera, Anastrepha  and  Rhagoletis  genera. 
     
     
         9 . A polynucleotide sequence according to  claim 8 , wherein one or both of the first and second splice control sequences is derived from  Ceratitis capitata.    
     
     
         10 . A polynucleotide sequence according to  claim 8 , wherein one of the first and second splice control sequences is derived from  Ceratitis capitata , and the other splice control sequence is derived from a different species. 
     
     
         11 . A polynucleotide sequence according to any one of the preceding claims, wherein one or both of the first and second gene expression systems further comprises an enhancer associated with the respective promoter, and at least one of the activating transcription factors acts at the respective enhancer. 
     
     
         12 . A polynucleotide sequence according to  claim 11 , wherein the enhancer, or one or both of the enhancers, is a tetO element comprising one or more repeats of tetO. 
     
     
         13 . A polynucleotide sequence according to  claim 12 , wherein the or each enhancer is, independently, tetO×7, tetO×14 or tetO×21. 
     
     
         14 . A polynucleotide sequence according to any one of the preceding claims, wherein one or both of the first and second promoters is expressed during at least embryonic development. 
     
     
         15 . A polynucleotide sequence according to any one of the preceding claims, wherein one or both of the first and second promoters is a minimal promoter. 
     
     
         16 . A polynucleotide sequence according to  claim 14  or  15 , wherein each of the first and second promoters is, independently, Hsp70 or sryα, preferably wherein one is Hsp70 and the other is sryα. 
     
     
         17 . A polynucleotide sequence according to any one of the preceding claims, wherein one of the first and second gene expression systems comprises a third dominant lethal gene and a third promoter 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. 
     
     
         18 . A polynucleotide sequence according to  claim 17 , as dependent on any one of  claims 11 - 13 , wherein said gene expression system further comprises an enhancer associated with the promoter of said gene expression system, and the third promoter is also associated with said enhancer, preferably wherein the promoter of said gene expression system is associated with one end of said enhancer and the third promoter is associated with the other end of said enhancer. 
     
     
         19 . A polynucleotide sequence according to  claim 17  or  18 , wherein the third dominant, lethal gene is VP16, and the third promoter is Hsp70 or sryα. 
     
     
         20 . A polynucleotide sequence according to any one of the preceding claims, wherein the polynucleotide sequence further comprises a genetic marker, preferably wherein the genetic marker is a fluorescent marker, more preferably wherein the genetic marker is DsRed2. 
     
     
         21 . A polynucleotide sequence according to any one of the preceding claims, 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 sryα 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 tetO×7,   the second gene expression system further comprises a second enhancer associated with the second promoter, wherein the second enhancer is tetO×14,   the polynucleotide sequence further comprises a third dominant lethal gene operably linked to a third promoter, the third promoter being associated 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 sequence further comprises a first genetic marker, which is DsRed2.   
     
     
         22 . A polynucleotide sequence according to any one of the preceding claims, wherein the polynucleotide sequence comprises the nucleotide sequence represented by SEQ ID NO: 1. 
     
     
         23 . A genetic construct for transforming an arthropod, comprising a polynucleotide sequence as described in any one of the preceding claims. 
     
     
         24 . A genetic construct according to  claim 23 , wherein the construct further comprises at least four transposon inverted repeats, forming at least two pairs of opposing transposon inverted repeats, wherein said polynucleotide sequence 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 sequence integrated in the host genome, without flanking transposon inverted repeats in the host genome. 
     
     
         25 . A genetic construct according to  claim 24 , wherein each of the transposon inverted repeats bounding said polynucleotide sequence is a minimal terminal inverted repeat, preferably wherein the transposon inverted repeats are from Class II transposable elements, more preferably piggyBac. 
     
     
         26 . A genetic construct according to  claim 24  or  25 , having four transposon inverted repeats. 
     
     
         27 . A genetic construct according to  claim 26 , wherein the 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 sequence and an external 5′ piggyBac end distal the polynucleotide sequence, and   the second pair consists of an internal 5′ piggyBac end proximal the polynucleotide sequence and an external 3′ piggyBac end distal the polynucleotide sequence, preferably wherein:   the internal 3′ piggyBac end consists of the polynucleotide sequence represented by SEQ ID NO: 32   the external 5′ piggyBac end consists of the polynucleotide sequence represented by SEQ ID NO:30   the internal 5′ piggyBac end consists of the polynucleotide sequence represented by SEQ ID NO: 30, and   the external 3′ piggyBac end consists of the polynucleotide sequence represented by SEQ ID NO: 31.   
     
     
         28 . A genetic construct according to any one of  claims 23 - 27 , further comprising at least one genetic marker between the transposon inverted repeats of at least one pair of opposing transposon inverted repeats, preferably comprising at least one genetic marker between the inverted repeats of each of the at least two pairs of opposing repeats. 
     
     
         29 . An arthropod comprising a polynucleotide sequence as described in any one of  claims 1 - 22 . 
     
     
         30 . An arthropod comprising the genetic construct as described in any one of  claims 23 - 28 . 
     
     
         31 . An arthropod according to  claim 29  or  30 , wherein the arthropod is a Tephritidae, Drosophilidae, Lonchaeidae, Pallopteridae, Platystomatidae, Pyrogotidae, Richardiidae or Ulidiidae, preferably a Tephritidae. 
     
     
         32 . An arthropod according to  claim 31 , wherein the arthropod is of the genus  Ceratitis , preferably being  Ceratitis capitata.    
     
     
         33 . An arthropod according to any one of  claims 30 - 32 , wherein the arthropod comprises the nucleotide sequence represented by SEQ ID NO: 2 or 3, preferably SEQ ID NO: 2. 
     
     
         34 . A method for detecting an arthropod as described in any one of  claims 30 - 33 , comprising the steps of:
 i) contacting a sample of DNA isolated from an insect with a primer pair specific for the transformed insect, wherein the first primer in the pair is specific for the polynucleotide sequence of the insect genetic construct and the second primer in the pair is specific for a genomic nucleotide sequence flanking the polynucleotide sequence,   ii) amplifying the sample of DNA, and   iii) detecting the amplified DNA.   
     
     
         35 . A method according to  claim 34  as dependent on  claim 33 , wherein the first primer has the nucleotide sequence represented by SEQ ID NO: 6 and the second primer has the nucleotide sequence represented by SEQ ID NO: 7. 
     
     
         36 . A method according to  claim 34  as dependent on  claim 33 , wherein the first primer has the nucleotide sequence represented by SEQ ID NO: 4 and the second primer has the nucleotide sequence represented by SEQ ID NO: 5. 
     
     
         37 . A method according to  claim 34  as dependent on  claim 33 , wherein the first primer has the nucleotide sequence represented by SEQ ID NO: 10 and the second primer has the nucleotide sequence represented by SEQ ID NO: 11. 
     
     
         38 . A method according to  claim 37 , wherein step i) further comprises contacting the sample of DNA with a probe specific to the PCR amplification product produced in step ii), for detection of the PCR amplification product, preferably wherein the probe is a dual labelled probe comprising a reporter molecule and a quencher molecule, and wherein the reporter molecule is released from the probe by Taq polymerase during PCR amplification, when said probe is annealed to said PCR amplification product. 
     
     
         39 . A method according to  claim 38 , wherein the probe has the nucleotide sequence represented by SEQ ID NO: 12. 
     
     
         40 . A primer for use in a method as described in  claim 34 , comprising the nucleotide sequence represented by one of SEQ ID NOs: 4, 5, 6, 7, 10 or 11. 
     
     
         41 . A primer for use in a method as described in  claim 38 , comprising the nucleotide sequence represented by SEQ ID NOs: 10 or 11. 
     
     
         42 . A probe for use in a method as described in  claim 38  or  39 , comprising the nucleotide sequence represented by SEQ ID NO: 12. 
     
     
         43 . A method of controlling an arthropod population comprising release of an arthropod as described in any one of  claims 29 - 33 , preferably wherein the arthropod is  Ceratitis capitata.