Abstract
Methods involving the release of transgenic insects in the field hold great promise for controlling vector-borne diseases and agricultural pests. Insect transformation depends on nonautonomous transposable elements as gene vectors. The resulting insertions are stable in the absence of suitable transposase, however, such absence cannot always be guaranteed. We describe a method for post-integration elimination of all transposon sequences in the pest insect Medfly, Ceratitis capitata. The resulting insertions lack transposon sequences and are therefore impervious to transposase activity.
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Acknowledgements
We thank Karen Clifton and staff at Oxitec for technical assistance, Nadia Pantic and Peng Gong for help at early stages of the project, Pedro Rendón for the Toliman Medfly strain and Tassos Mintzas, Sinead O'Connell and Derric Nimmo for advice and critical review of the manuscript. This work was funded in part by the UK Biotechnology and Biological Sciences Research Council.
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Contributions
T.H.D. and G.C.C. contributed equally to this work. T.H.D., C.E.P. and L.J. designed and constructed the DNA constructs and assisted with the molecular analysis of transgenics. G.C.C. and K.C.C. created the transgenic Medflies and conducted most of the phenotypic and molecular analysis. N.I.M., M.J.E. and G.F. performed preliminary experiments and developed the marker system. L.A. conceived and supervised the project and wrote the paper with G.C.C. All authors discussed the results and commented on the manuscript.
Note: Supplementary information is available on the Nature Biotechnology website.
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Those authors affiliated with Oxitec Ltd. are employees or students of this company. Oxitec supplied salary and other support for the research program. Also, some of these authors have shares or share options in Oxitec Ltd. Both Oxitec Ltd. and Oxford University have one or more patents or patent applications related to the subject of this paper.
Supplementary information
Supplementary Fig. 1
piggyBac-based constructs used in this paper. (PDF 121 kb)
Supplementary Fig. 2
Sequence and location of primers used to analyse insertions. (PDF 115 kb)
Supplementary Fig. 3
Expression of CopGreen, PhiYFP and J-Red fluorescent markers. (PDF 125 kb)
Supplementary Table 1
Post-integration removal of piggyBac ends. (PDF 97 kb)
Supplementary Table 2
Mobilization rates of an X-linked composite insertion. (PDF 94 kb)
Supplementary Table 3
Mobilization rates of integrated transposons. (PDF 116 kb)
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Dafa'alla, T., Condon, G., Condon, K. et al. Transposon-free insertions for insect genetic engineering. Nat Biotechnol 24, 820–821 (2006). https://doi.org/10.1038/nbt1221
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DOI: https://doi.org/10.1038/nbt1221
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