Biocontrol

Abstract

Provided is an arthropod male germline gene expression system suitable for conditional expression of an effector gene in an Arthropod male germline. The system comprises a first expression unit comprising an effector gene and a promoter therefor operably linked thereto; and a second expression unit. Said second unit comprises a coding sequence for a transcription factor and an upstream regulatory element operably linked thereto, the transcription factor being capable of acting upon the promoter in the first expression unit to drive expression of the effector gene. The upstream regulatory element includes a promoter for the transcription factor; and a 5′ UTR adjacent a start site for the transcription factor coding sequence. The upstream regulatory element driving sufficient expression of the transcription factor such that the transcription factor protein in turn drives transcription of the effector gene before meiosis. Also provided are uses of the system for instance in methods of biocontrol and quality control.

1 ) An arthropod male germline gene expression system suitable for conditional expression of an effector gene in an Arthropod male germline, the system comprising:
 a first expression unit comprising an effector gene and a promoter therefor operably linked thereto;   a second expression unit comprising a coding sequence for a transcription factor and an upstream regulatory element operably linked thereto, the transcription factor being capable of acting upon the promoter in the first expression unit to drive expression of the effector gene, the upstream regulatory element including:
 a promoter for the transcription factor; and 
 a 5′ UTR adjacent a start site for the transcription factor coding sequence; 
   
       the upstream regulatory element driving sufficient expression of the transcription factor such that the transcription factor protein in turn drives transcription of the effector gene before meiosis. 
     
     
         2 ) A gene expression system according to  claim 1 , wherein the transcription factor is a transcriptional activator, such as tTA, GAL4 or their variants. 
     
     
         3 ) A gene expression system according to any preceding claim, wherein the effector is endonuclease, most preferably a 3-Zn finger nuclease. 
     
     
         4 ) A gene expression system according to any preceding claim, wherein the promoter of the first expression unit is a minimal promoter. 
     
     
         5 ) A gene expression system according to any preceding claim, wherein the promoter of the upstream regulatory element in the second expression unit is most from topi, aly or Beta-2 Tubulin (B2T) or homologues thereof. 
     
     
         6 ) A gene expression system according to any preceding claim, wherein the 5′ UTR in the upstream regulatory element of the second expression unit is that from hsp83, preferably from Medfly or homologues of hsp83, particularly the homologue of hsp83 found in the target arthropod. 
     
     
         7 ) A gene expression system according to any preceding claim, wherein the Gal4-UAS system is used, such that the transcription factor is GAL4. 
     
     
         8 ) A gene expression system according to any of  claims 1 - 6 , wherein the system is an inducible system, where induction occurs by provision of a chemical entity, such a tetracycline or one of its analogues including doxycycline. 
     
     
         9 ) A gene expression system according to  claim 8 , wherein the transcription factor in the second expression unit is tTA or a variant thereof (tTAV, tTAV2, tTAV3). 
     
     
         10 ) A gene expression system according to any preceding claim, wherein the transcription factor of the second expression unit is tTA or a variant and the first expression unit includes the tet operator (tetO). 
     
     
         11 ) A gene expression system according to any preceding claim, wherein the arthropod is an insect, preferably a Dipteran, including a Tephritid, such as a Medfly or Olive fly. 
     
     
         12 ) A gene expression system according to any preceding claim, wherein the effector is a nuclease and confers or imparts paternal effect lethality. 
     
     
         13 ) A method of expressing an effector protein in a gonad or sperm, comprising transforming the gonad with the expression system according to any preceding claim. 
     
     
         14 ) A method of population control comprising expressing the effector protein of the expression system according to any of  claims 1 - 12  in the gonads of a male arthropod. 
     
     
         15 ) A method of resistance management comprising use of the expression system according to any of  claims 1 - 12 . 
     
     
         16 ) A method of quality control, comprising including a reporter, such as a fluorescent protein as, or in addition to, the effector protein in the expression system according to any of  claims 1 - 12 , such that individuals where expression from the system has been induced or de-repressed become visible under suitable wavelengths of light. 
     
     
         17 ) A method of determining the mating status of a female arthropod, comprising use of the expression system according to any of  claims 1 - 12  in a transgenic male population, wherein said system comprises a marker such as a fluorescent reporter protein; and where sperm is present, assaying for the presence of said marker in a female; the presence of the marker being indicative that the female has mated with a transgenic male carrying the system.