Transposon-free insertions for insect genetic engineering

Dafa'alla, Tarig; Condon, George C.; Condon, Kirsty C; Phillips, Caroline E.; Morrison, Neil I.; Jin, Li; Epton, Matthew J; Fu, Guoliang; Alphey, Luke

doi:10.1038/nbt1221

Cited by 59 publications

(66 citation statements)

References 13 publications

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“…This obviates serious programmatic and ecological concerns for transgene instability, which would initially result in loss of desired transgene phenotypes, and then potentially, horizontal transmission of the transgene into unintended host species. The first mexfly transformation study also stabilized vectors, but by re-mobilizing both terminal sequences resulting in transposon-free transgene insertions (Condon et al 2007b;Dafa'alla et al 2006). However, these stabilized transgenes are more difficult to achieve, require another marker gene, and the practical need for post-integration deletion of both termini is not compelling.…”

Section: Discussionmentioning

confidence: 97%

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

et al. 2010

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The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13-21% per fertile G(0) individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3' terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5-10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.

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Section: Discussionmentioning

confidence: 97%

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

et al. 2010

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“…In these instances stability of the integrated transgene, such as the testis-speciWc 2tubulin-egfp transgene used for the separation of females from males in diVerent mosquito species (Catteruccia et al 2005;Smith et al 2007), would be an essential requisite in SIT programs where large numbers of mosquitoes need to be reared, sexed and released. Indeed a series of systems have been developed in Drosophila and other fruit Xies to achieve the stabilization of integrated transposons by removing one or both terminal inverted repeats (Handler et al 2004;Dafa'alla et al 2006). However, these systems require the development of a large number of transgenic lines and a high eYciency of remobilization of the transposon in use, neither of which at present represent a viable option in Anopheles or other mosquitoes.…”

Section: Discussionmentioning

confidence: 99%

Post-integration behavior of a Minos transposon in the malaria mosquito Anopheles stephensi

et al. 2007

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Transposable elements represent important tools to perform functional studies in insects. In Drosophila melanogaster, the remobilization properties of transposable elements have been utilized for enhancer-trapping and insertional mutagenesis experiments, which have considerably helped in the functional characterization of the fruitfly genome. In Anopheles mosquitoes, the sole vectors of human malaria, as well as in other mosquito vectors of disease, the use of transposons has also been advocated to achieve the spread of anti-parasitic genes throughout field populations. Here we report on the post-integration behavior of the Minos transposon in both the germ-line and somatic tissues of Anopheles mosquitoes. Transgenic An. stephensi lines developed using the piggyBac transposon and expressing the Minos transposase were tested for their ability to remobilize an X-linked Minos element. Germ-line remobilization events were not detected, while somatic excisions and transpositions were consistently recovered. The analysis of these events showed that Minos activity in Anopheles cells is characterized by unconventional functionality of the transposon. In the two cases analyzed, re-integration of the transposon occurred onto the same X chromosome, suggesting a tendency for local hopping of Minos in the mosquito genome. This is the first report of the post-integration behavior of a transposable element in a human malaria vector.

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“…The consequence of such transgene-transposase exposure could be the remobilization of a transgene to another genomic location or total loss of a transgene from an insect's genome. Measures to avoid potential transgene remobilization in engineered dipterans such as postintegrational transgene modification to alter the transposon and achieve nonmobilization or stability has been demonstrated in D. melanogaster and C. capitata [86][87][88]. Other strategies that offer transgene stability are becoming available.…”

Section: Limitations Of Transgenic Technologymentioning

confidence: 99%

Developing the Arsenal Against Pest and Vector Dipterans: Inputs of Transgenic and Paratransgenic Biotechnologies

Ogaugwu¹,

Durvasula²

2017

Biological Control of Pest and Vector Insects

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Insects are the most numerous of all animals and are found in almost every inhabitable place on earth. The order Diptera (true flies) contains many members that are notorious agricultural pests, nuisance or vectors of diseases. The list is long: mosquitoes, tsetse flies, screw worms, fruit flies, sand flies, blow flies, house flies, gall and biting midges, black flies, leaf miners, horse flies, and so on. Efforts to combat some of these pests and vectors have resulted in control measures such as the chemical, physical, and cultural control methods. These methods, though largely beneficial, have disadvantages and limitations, which sometimes seem to outweigh the problems initially sought to be controlled. The chemical method, for example, is not environment-friendly since it negatively affects many nontarget organisms and disrupts ecosystem balance. Development of insecticide resistance by pests/vectors is another concern. Molecular biotechnology has introduced vast arrays of novel ways to tackle pests and disease vectors, as well as improve the potency of existing control methods. This chapter looks at transgenic and paratransgenic biotechnologies and how they have been applied so far to develop and expand the arsenal against dipteran pests and disease vectors. Further, we discuss the advantages, disadvantages, and limitations of these technologies.

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Transposon-free insertions for insect genetic engineering

Cited by 59 publications

References 13 publications

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

Post-integration behavior of a Minos transposon in the malaria mosquito Anopheles stephensi

Developing the Arsenal Against Pest and Vector Dipterans: Inputs of Transgenic and Paratransgenic Biotechnologies

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