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Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches.

Eckermann KN, Dippel S, KaramiNejadRanjbar M, Ahmed HM, Curril IM, Wimmer EA - BMC Genet. (2014)

Bottom Line: However, to create 'redundant killing' an additional independent conditional expression system is required.We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

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ABSTRACT

Background: The Sterile Insect Technique (SIT) is an accepted species-specific genetic control approach that acts as an insect birth control measure, which can be improved by biotechnological engineering to facilitate its use and widen its applicability. First transgenic insects carrying a single killing system have already been released in small scale trials. However, to evade resistance development to such transgenic approaches, completely independent ways of transgenic killing should be established and combined.

Perspective: Most established transgenic sexing and reproductive sterility systems are based on the binary tTA expression system that can be suppressed by adding tetracycline to the food. However, to create 'redundant killing' an additional independent conditional expression system is required. Here we present a perspective on the use of a second food-controllable binary expression system - the inducible Q system - that could be used in combination with site-specific recombinases to generate independent transgenic killing systems. We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.

Conclusion: A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

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Sexing using female-specific splicing under the control of the repressible tTA-system. The depicted transgenic sexing system [41,42] uses a sex-specifically spliced intron and a hyperactive pro-apoptotic gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator (tTA). To cause early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter. During rearing of such strains, addition of tetracycline (TET) to the food keeps the system in the OFF state, as tetracycline blocks the binding of tTA to its response element (TRE). For the release generation, tetracycline is absent in the food and therefore the sexing system is ON: in males, the male specific splicing of the transformer intron (tra-I) - placed after the translation start codon (ATG) of the effector gene - includes a small exon containing a TAA stop codon between the start codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.
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Figure 1: Sexing using female-specific splicing under the control of the repressible tTA-system. The depicted transgenic sexing system [41,42] uses a sex-specifically spliced intron and a hyperactive pro-apoptotic gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator (tTA). To cause early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter. During rearing of such strains, addition of tetracycline (TET) to the food keeps the system in the OFF state, as tetracycline blocks the binding of tTA to its response element (TRE). For the release generation, tetracycline is absent in the food and therefore the sexing system is ON: in males, the male specific splicing of the transformer intron (tra-I) - placed after the translation start codon (ATG) of the effector gene - includes a small exon containing a TAA stop codon between the start codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.

Mentions: To generate transgenic sexing systems, female lethality was first developed and tested in D. melanogaster and based on the female-specific expression of conditional lethal genes [35,36]. More recently transgenic sexing systems for tephritid fruit flies have been generated using a female-specifically spliced intron from the transformer gene. First it was used in an autocidal expression loop with the female lethality occurring at late larval stages in the Medfly Ceratitis capitata [37]. This system has successfully been transferred to other Tephritids such as the olive fly Bactrocera oleae [38] and also to blowflies [39]- devastating pests of livestock - as well as to lepidopterans [40]. Furthermore, embryonic transgenic sexing systems have combined the use of such a female-specifically spliced intron with an early embryonic expression mediated by cis-regulatory elements from early acting cellularization genes that indirectly and controllably drive the expression of a hyper-active pro-apoptotic gene (Figure 1) [41,42]. An even better understanding of the sex differentiation pathways in insects will provide us with additional strategies for synthetic genetic-based tools for large scale sex separation in SIT applications [43] based on either female killing or actual female sex-reversal [44,45].


Perspective on the combined use of an independent transgenic sexing and a multifactorial reproductive sterility system to avoid resistance development against transgenic Sterile Insect Technique approaches.

Eckermann KN, Dippel S, KaramiNejadRanjbar M, Ahmed HM, Curril IM, Wimmer EA - BMC Genet. (2014)

Sexing using female-specific splicing under the control of the repressible tTA-system. The depicted transgenic sexing system [41,42] uses a sex-specifically spliced intron and a hyperactive pro-apoptotic gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator (tTA). To cause early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter. During rearing of such strains, addition of tetracycline (TET) to the food keeps the system in the OFF state, as tetracycline blocks the binding of tTA to its response element (TRE). For the release generation, tetracycline is absent in the food and therefore the sexing system is ON: in males, the male specific splicing of the transformer intron (tra-I) - placed after the translation start codon (ATG) of the effector gene - includes a small exon containing a TAA stop codon between the start codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255789&req=5

Figure 1: Sexing using female-specific splicing under the control of the repressible tTA-system. The depicted transgenic sexing system [41,42] uses a sex-specifically spliced intron and a hyperactive pro-apoptotic gene to generate female-specific lethality under the regulation of the tetracycline-controlled transactivator (tTA). To cause early embryonic lethality and thus avoidance of larval survival, the tTA is under the control of an early embryonic promoter. During rearing of such strains, addition of tetracycline (TET) to the food keeps the system in the OFF state, as tetracycline blocks the binding of tTA to its response element (TRE). For the release generation, tetracycline is absent in the food and therefore the sexing system is ON: in males, the male specific splicing of the transformer intron (tra-I) - placed after the translation start codon (ATG) of the effector gene - includes a small exon containing a TAA stop codon between the start codon and the rest of the effector gene and therefore prevents the production of the functional pro-apoptotic effector protein allowing the males to survive; whereas in the females the female specific splicing of the tra-I produces a functional effector and the embryonic cells are driven into apoptosis, which leads to female-specific embryonic lethality.
Mentions: To generate transgenic sexing systems, female lethality was first developed and tested in D. melanogaster and based on the female-specific expression of conditional lethal genes [35,36]. More recently transgenic sexing systems for tephritid fruit flies have been generated using a female-specifically spliced intron from the transformer gene. First it was used in an autocidal expression loop with the female lethality occurring at late larval stages in the Medfly Ceratitis capitata [37]. This system has successfully been transferred to other Tephritids such as the olive fly Bactrocera oleae [38] and also to blowflies [39]- devastating pests of livestock - as well as to lepidopterans [40]. Furthermore, embryonic transgenic sexing systems have combined the use of such a female-specifically spliced intron with an early embryonic expression mediated by cis-regulatory elements from early acting cellularization genes that indirectly and controllably drive the expression of a hyper-active pro-apoptotic gene (Figure 1) [41,42]. An even better understanding of the sex differentiation pathways in insects will provide us with additional strategies for synthetic genetic-based tools for large scale sex separation in SIT applications [43] based on either female killing or actual female sex-reversal [44,45].

Bottom Line: However, to create 'redundant killing' an additional independent conditional expression system is required.We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The Sterile Insect Technique (SIT) is an accepted species-specific genetic control approach that acts as an insect birth control measure, which can be improved by biotechnological engineering to facilitate its use and widen its applicability. First transgenic insects carrying a single killing system have already been released in small scale trials. However, to evade resistance development to such transgenic approaches, completely independent ways of transgenic killing should be established and combined.

Perspective: Most established transgenic sexing and reproductive sterility systems are based on the binary tTA expression system that can be suppressed by adding tetracycline to the food. However, to create 'redundant killing' an additional independent conditional expression system is required. Here we present a perspective on the use of a second food-controllable binary expression system - the inducible Q system - that could be used in combination with site-specific recombinases to generate independent transgenic killing systems. We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding.

Conclusion: A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT.

Show MeSH
Related in: MedlinePlus