Gene expression system
AssigneeOxitec Limited
InventorsMartha KOUKIDOU, Luke ALPHEY, Simon WARNER
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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.
Claims43
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
A genetic construct for transforming an arthropod, comprising a polynucleotide sequence as described in any one of the preceding claims.
Dependencies are inferred from the claim text.