Limits...
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.

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

Rearing scheme for combined female lethality and multifactorial reproductive sterility systems. A Under regular rearing conditions, tetracycline (TET) is added to the food to repress the female lethality, quinic acid (QA) is not required for rearing. B The adult parents of the release generation will be changed to food without TET, still also without QA. This is necessary to avoid suppression of the early embryonic lethality in the next generation by maternally transferred TET to the oocyte. The female lethality system is still off, since the early embryonic promoter is not driving tTA at adult stages. C The release generation is then reared on food without TET but with added QA. Due to the lack of TET the female lethality system is switched on and the females die during early development. The QA leads to the activation of the Q system that leads to the expression of a site specific recombinase, which in turn mediates the spermatogenesis-specific expression of the Cas9 endonuclease by removing a recombination site-flanked spacer cassette. D The released males (no TET, no further QA) express high levels of the endonuclease Cas9 and multiple guide RNAs during spermatogenesis causing shredded chromosomes that will lead to lethal aneuploidy in the next generation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4255789&req=5

Figure 4: Rearing scheme for combined female lethality and multifactorial reproductive sterility systems. A Under regular rearing conditions, tetracycline (TET) is added to the food to repress the female lethality, quinic acid (QA) is not required for rearing. B The adult parents of the release generation will be changed to food without TET, still also without QA. This is necessary to avoid suppression of the early embryonic lethality in the next generation by maternally transferred TET to the oocyte. The female lethality system is still off, since the early embryonic promoter is not driving tTA at adult stages. C The release generation is then reared on food without TET but with added QA. Due to the lack of TET the female lethality system is switched on and the females die during early development. The QA leads to the activation of the Q system that leads to the expression of a site specific recombinase, which in turn mediates the spermatogenesis-specific expression of the Cas9 endonuclease by removing a recombination site-flanked spacer cassette. D The released males (no TET, no further QA) express high levels of the endonuclease Cas9 and multiple guide RNAs during spermatogenesis causing shredded chromosomes that will lead to lethal aneuploidy in the next generation.

Mentions: The insect strains carrying the combined transgenic female lethality and multifactorial reproductive male sterility systems would be reared on tetracycline containing food to suppress the female-specific lethality. The male reproductive sterility would not be activated yet, since the repressor QS would keep the system in an OFF state (Figure 4A). The adult flies of the pre-release generation would then be aged on tetracycline-free food (Figure 4B) in order to stop the suppression of the embryonic female-specific lethality in the next generation [29,41,42]. The release generation should then be grown also on tetracycline-free larval food in order to keep the embryonic sexing system on to produce males only: in the absence of tetracycline, the synthetic transactivator tTA would activate a hyper-active pro-apoptotic gene that would lead to programmed cell death in the female embryos, as only the female-specific splicing of the transformer intron in this transcript results in the production of an mRNA capable of translating the functional hyper-active pro-apoptotic protein (Figure 4C). The larval food for the release generation would, however, need to contain quinic acid to inactivate the repressor QS, which would then allow the activator QF to induce the expression of the flp recombinase gene, which then in turn would remove the Q system regulators and mediate the expression of the heterologous endonuclease Cas9 during spermatogenesis (Figure 4C). Released males (Figure 4D) would produce sperm with shredded chromosomes leading to lethal aneuploidy in the next generation similar to radiation-induced reproductive sterility without suffering of somatic damages that cause reduced fitness.


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)

Rearing scheme for combined female lethality and multifactorial reproductive sterility systems. A Under regular rearing conditions, tetracycline (TET) is added to the food to repress the female lethality, quinic acid (QA) is not required for rearing. B The adult parents of the release generation will be changed to food without TET, still also without QA. This is necessary to avoid suppression of the early embryonic lethality in the next generation by maternally transferred TET to the oocyte. The female lethality system is still off, since the early embryonic promoter is not driving tTA at adult stages. C The release generation is then reared on food without TET but with added QA. Due to the lack of TET the female lethality system is switched on and the females die during early development. The QA leads to the activation of the Q system that leads to the expression of a site specific recombinase, which in turn mediates the spermatogenesis-specific expression of the Cas9 endonuclease by removing a recombination site-flanked spacer cassette. D The released males (no TET, no further QA) express high levels of the endonuclease Cas9 and multiple guide RNAs during spermatogenesis causing shredded chromosomes that will lead to lethal aneuploidy in the next generation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Rearing scheme for combined female lethality and multifactorial reproductive sterility systems. A Under regular rearing conditions, tetracycline (TET) is added to the food to repress the female lethality, quinic acid (QA) is not required for rearing. B The adult parents of the release generation will be changed to food without TET, still also without QA. This is necessary to avoid suppression of the early embryonic lethality in the next generation by maternally transferred TET to the oocyte. The female lethality system is still off, since the early embryonic promoter is not driving tTA at adult stages. C The release generation is then reared on food without TET but with added QA. Due to the lack of TET the female lethality system is switched on and the females die during early development. The QA leads to the activation of the Q system that leads to the expression of a site specific recombinase, which in turn mediates the spermatogenesis-specific expression of the Cas9 endonuclease by removing a recombination site-flanked spacer cassette. D The released males (no TET, no further QA) express high levels of the endonuclease Cas9 and multiple guide RNAs during spermatogenesis causing shredded chromosomes that will lead to lethal aneuploidy in the next generation.
Mentions: The insect strains carrying the combined transgenic female lethality and multifactorial reproductive male sterility systems would be reared on tetracycline containing food to suppress the female-specific lethality. The male reproductive sterility would not be activated yet, since the repressor QS would keep the system in an OFF state (Figure 4A). The adult flies of the pre-release generation would then be aged on tetracycline-free food (Figure 4B) in order to stop the suppression of the embryonic female-specific lethality in the next generation [29,41,42]. The release generation should then be grown also on tetracycline-free larval food in order to keep the embryonic sexing system on to produce males only: in the absence of tetracycline, the synthetic transactivator tTA would activate a hyper-active pro-apoptotic gene that would lead to programmed cell death in the female embryos, as only the female-specific splicing of the transformer intron in this transcript results in the production of an mRNA capable of translating the functional hyper-active pro-apoptotic protein (Figure 4C). The larval food for the release generation would, however, need to contain quinic acid to inactivate the repressor QS, which would then allow the activator QF to induce the expression of the flp recombinase gene, which then in turn would remove the Q system regulators and mediate the expression of the heterologous endonuclease Cas9 during spermatogenesis (Figure 4C). Released males (Figure 4D) would produce sperm with shredded chromosomes leading to lethal aneuploidy in the next generation similar to radiation-induced reproductive sterility without suffering of somatic damages that cause reduced fitness.

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