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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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Reproductive sterility using a homing endonuclease controlled by the inducible Q-system in combination with site-specific recombination. The proposed reproductive sterility system is based on the inducible binary expression system Q [48], in which quinic acid (QA) acts as an inducer that hinders the repressor QS from complexing the transcriptional activator QF, which can activate its target genes by binding to a Q upstream activation sequence (QUAS). To generate male reproductive sterility systems the spermatogenesis-specific promoter of the β2 tubulin (β2t) gene can be suitably used to affect either the sperm itself or the progeny sired by the sperm. The Q system can be combined with a recombinase mediated transcription regulation system to render the induction of an effector gene expression permanent and independent of the presence of the inducer QA. In this dual system, QF drives the expression of a site-specific recombinase (FLP) that can in turn remove a flp-out cassette [57], which contains a transcriptional terminator (SV40) and is flanked by flp recombinant target sites (FRTs) in direct orientation. After the removal of the transcriptional terminator, the directed expression of an effector gene is mediated by the tissue-specific promoter 5' to the FRT. Since the Q system components are superfluous after the activation of the effector gene, they can also be placed into the flp-out cassette. To make sure that both components of the Q system are translated in a bi-cistronic messenger RNA, they will be separated by an internal ribosome entry site (IRES). A homing endonuclease targeting the progeny genome can be employed as an effector that would kill the progeny but not the sperm itself [34]. During regular rearing this male reproductive sterility would be kept in an OFF state, as at the absence of QA the repressor QS will mask QF and block its activation potential. Only after the addition of QA to the food in the release generation, QS will be inactivated and QF thereby allowed to activate the expression of the flp recombinase (FLP), which in turn would remove the Q system regulators and at the same time mediate the expression of the homing endonuclease that could block development of the next generation and thus cause male reproductive sterility.
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Figure 2: Reproductive sterility using a homing endonuclease controlled by the inducible Q-system in combination with site-specific recombination. The proposed reproductive sterility system is based on the inducible binary expression system Q [48], in which quinic acid (QA) acts as an inducer that hinders the repressor QS from complexing the transcriptional activator QF, which can activate its target genes by binding to a Q upstream activation sequence (QUAS). To generate male reproductive sterility systems the spermatogenesis-specific promoter of the β2 tubulin (β2t) gene can be suitably used to affect either the sperm itself or the progeny sired by the sperm. The Q system can be combined with a recombinase mediated transcription regulation system to render the induction of an effector gene expression permanent and independent of the presence of the inducer QA. In this dual system, QF drives the expression of a site-specific recombinase (FLP) that can in turn remove a flp-out cassette [57], which contains a transcriptional terminator (SV40) and is flanked by flp recombinant target sites (FRTs) in direct orientation. After the removal of the transcriptional terminator, the directed expression of an effector gene is mediated by the tissue-specific promoter 5' to the FRT. Since the Q system components are superfluous after the activation of the effector gene, they can also be placed into the flp-out cassette. To make sure that both components of the Q system are translated in a bi-cistronic messenger RNA, they will be separated by an internal ribosome entry site (IRES). A homing endonuclease targeting the progeny genome can be employed as an effector that would kill the progeny but not the sperm itself [34]. During regular rearing this male reproductive sterility would be kept in an OFF state, as at the absence of QA the repressor QS will mask QF and block its activation potential. Only after the addition of QA to the food in the release generation, QS will be inactivated and QF thereby allowed to activate the expression of the flp recombinase (FLP), which in turn would remove the Q system regulators and at the same time mediate the expression of the homing endonuclease that could block development of the next generation and thus cause male reproductive sterility.

Mentions: Recently a second food-additive controllable expression system - the Q system - has been shown to work ex vivo in mammalian cells as well as in vivo in the vinegar fly D. melanogaster [48,49]. The broad applicability of this system is also demonstrated by its functionality in the nematode worm Caenorhabditis elegans [50]. The Q system is based on the regulatory genes of the gene cluster qa from the bread mold Neurospora crassa, which allows the fungus to utilize quinic acid as a carbon source [51]. Quinic acid can be found in high concentrations both in herbaceous plants as well as conifers [52] and at especially high levels in unripe fruits [53]. Several molds are able to use quinic acid as carbon source and have specific gene clusters for the catabolic pathway [54]. The regulatory genes of the cluster ensure that the catabolic enzymes are only expressed at the presence of quinic acid: one gene, qa-1F (QF), acts as DNA-binding transcriptional activator of all cluster genes, whereas another regulatory gene, qa-1S (QS), acts as a repressor that does not bind DNA itself but inactivates the activator QF by complex formation [54]. Quinic acid acts as an inducer by hindering the repressor QS from complexing QF, which then can activate its target genes (Figure 2). Therefore, the Q system is actually an inducible binary expression system with the food additive, quinic acid, leading to the activation of controlled gene expression. This and the fact that quinic acid is found widespread in nature [52,53] do not allow us to use this system in an analogous way to the tTA system. However, it offers a completely independent food additive-controlled expression system that should be utilized for novel transgenic SIT approaches.


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)

Reproductive sterility using a homing endonuclease controlled by the inducible Q-system in combination with site-specific recombination. The proposed reproductive sterility system is based on the inducible binary expression system Q [48], in which quinic acid (QA) acts as an inducer that hinders the repressor QS from complexing the transcriptional activator QF, which can activate its target genes by binding to a Q upstream activation sequence (QUAS). To generate male reproductive sterility systems the spermatogenesis-specific promoter of the β2 tubulin (β2t) gene can be suitably used to affect either the sperm itself or the progeny sired by the sperm. The Q system can be combined with a recombinase mediated transcription regulation system to render the induction of an effector gene expression permanent and independent of the presence of the inducer QA. In this dual system, QF drives the expression of a site-specific recombinase (FLP) that can in turn remove a flp-out cassette [57], which contains a transcriptional terminator (SV40) and is flanked by flp recombinant target sites (FRTs) in direct orientation. After the removal of the transcriptional terminator, the directed expression of an effector gene is mediated by the tissue-specific promoter 5' to the FRT. Since the Q system components are superfluous after the activation of the effector gene, they can also be placed into the flp-out cassette. To make sure that both components of the Q system are translated in a bi-cistronic messenger RNA, they will be separated by an internal ribosome entry site (IRES). A homing endonuclease targeting the progeny genome can be employed as an effector that would kill the progeny but not the sperm itself [34]. During regular rearing this male reproductive sterility would be kept in an OFF state, as at the absence of QA the repressor QS will mask QF and block its activation potential. Only after the addition of QA to the food in the release generation, QS will be inactivated and QF thereby allowed to activate the expression of the flp recombinase (FLP), which in turn would remove the Q system regulators and at the same time mediate the expression of the homing endonuclease that could block development of the next generation and thus cause male reproductive sterility.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Reproductive sterility using a homing endonuclease controlled by the inducible Q-system in combination with site-specific recombination. The proposed reproductive sterility system is based on the inducible binary expression system Q [48], in which quinic acid (QA) acts as an inducer that hinders the repressor QS from complexing the transcriptional activator QF, which can activate its target genes by binding to a Q upstream activation sequence (QUAS). To generate male reproductive sterility systems the spermatogenesis-specific promoter of the β2 tubulin (β2t) gene can be suitably used to affect either the sperm itself or the progeny sired by the sperm. The Q system can be combined with a recombinase mediated transcription regulation system to render the induction of an effector gene expression permanent and independent of the presence of the inducer QA. In this dual system, QF drives the expression of a site-specific recombinase (FLP) that can in turn remove a flp-out cassette [57], which contains a transcriptional terminator (SV40) and is flanked by flp recombinant target sites (FRTs) in direct orientation. After the removal of the transcriptional terminator, the directed expression of an effector gene is mediated by the tissue-specific promoter 5' to the FRT. Since the Q system components are superfluous after the activation of the effector gene, they can also be placed into the flp-out cassette. To make sure that both components of the Q system are translated in a bi-cistronic messenger RNA, they will be separated by an internal ribosome entry site (IRES). A homing endonuclease targeting the progeny genome can be employed as an effector that would kill the progeny but not the sperm itself [34]. During regular rearing this male reproductive sterility would be kept in an OFF state, as at the absence of QA the repressor QS will mask QF and block its activation potential. Only after the addition of QA to the food in the release generation, QS will be inactivated and QF thereby allowed to activate the expression of the flp recombinase (FLP), which in turn would remove the Q system regulators and at the same time mediate the expression of the homing endonuclease that could block development of the next generation and thus cause male reproductive sterility.
Mentions: Recently a second food-additive controllable expression system - the Q system - has been shown to work ex vivo in mammalian cells as well as in vivo in the vinegar fly D. melanogaster [48,49]. The broad applicability of this system is also demonstrated by its functionality in the nematode worm Caenorhabditis elegans [50]. The Q system is based on the regulatory genes of the gene cluster qa from the bread mold Neurospora crassa, which allows the fungus to utilize quinic acid as a carbon source [51]. Quinic acid can be found in high concentrations both in herbaceous plants as well as conifers [52] and at especially high levels in unripe fruits [53]. Several molds are able to use quinic acid as carbon source and have specific gene clusters for the catabolic pathway [54]. The regulatory genes of the cluster ensure that the catabolic enzymes are only expressed at the presence of quinic acid: one gene, qa-1F (QF), acts as DNA-binding transcriptional activator of all cluster genes, whereas another regulatory gene, qa-1S (QS), acts as a repressor that does not bind DNA itself but inactivates the activator QF by complex formation [54]. Quinic acid acts as an inducer by hindering the repressor QS from complexing QF, which then can activate its target genes (Figure 2). Therefore, the Q system is actually an inducible binary expression system with the food additive, quinic acid, leading to the activation of controlled gene expression. This and the fact that quinic acid is found widespread in nature [52,53] do not allow us to use this system in an analogous way to the tTA system. However, it offers a completely independent food additive-controlled expression system that should be utilized for novel transgenic SIT approaches.

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