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Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae

Nature Protocols volume 9, pages 1698–1712 (2014)Cite this article

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Abstract

Current transgenic methodology developed for mosquitoes has not been applied widely to the major malaria vector Anopheles gambiae, which has proved more difficult to genetically manipulate than other mosquito species and dipteran insects. In this protocol, we describe ΦC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The ΦC31 system has many advantages over 'classical' transposon-mediated germline transformation systems, because it allows integration of large transgenes at specific, characterized genomic locations. Starting from a general protocol, we have optimized steps from embryo collection to co-injection of transgene-containing plasmid and in vitro–produced ΦC31 integrase mRNA. We also provide tips for screening transgenic larvae. The outlined procedure provides robust transformation in A. gambiae, resulting in homozygous transgenic lines in ∼2–3 months.

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Figure 1: Schematic representation of ΦC31 site-specific transformation in A. gambiae.
Figure 2: Fluorescence profile of larvae from standard docking strain and self-docking strain.
Figure 3: Timeline and flow diagram for site-specific ΦC31 transformation in A. gambiae mosquitoes.
Figure 4: Oviposition chamber.
Figure 5: G1 screening system.
Figure 6: G0 larvae expressing transient fluorescence.
Figure 7: False-positive G1 larvae.

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DDBJ/GenBank/EMBL

European Nucleotide Archive

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Acknowledgements

We are grateful to the Photography Department of Institut Pasteur, especially F. Gardy and J.-M. Panaud for the production of the video for embryo alignment, and to M. Calos for pET11-ΦC31 and original attP and attB constructs. We thank A. James, A. Handler and N. Windbichler for helpful technical discussions. Support to E.P. was from an ANR-07-MIME-O25-01 award to C.B., from the Roux Foundation (Pasteur Institute) and from a UE323173 Anopath award to K.D.V.; to C.B. from award no. ANR-10-LABX-62-IBEID; to P.E. from a Wellcome Trust Programme grant (0084582) and the EU FP7 (INFRAVEC); and to G.J.L. from European Commission grant no. 223726 (TransMalariaBloc) and a Biotechnology and Biological Sciences Research Council grant (no. BB/F021933/1).

Author information

Author notes

  1. Gareth J Lycett, Paul Eggleston and Catherine Bourgouin: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Pasteur, Genetics and Genomics of Insect Vectors, CNRS unit URA3012, Paris, France

    Emilie Pondeville, Nicolas Puchot, Kenneth D Vernick & Catherine Bourgouin

  2. Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK

    Janet M Meredith & Paul Eggleston

  3. Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK

    Amy Lynd & Gareth J Lycett

Authors
  1. Emilie Pondeville
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Contributions

E.P., N.P., J.M.M., A.L. and G.J.L. performed the research; C.B., G.J.L. and P.E. designed and supervised the research; K.D.V. supervised the research; and E.P., A.L., G.J.L., P.E. and C.B. wrote the manuscript.

Corresponding authors

Correspondence to Emilie Pondeville or Catherine Bourgouin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Primers and PCR parameters to detect transgene site-specific integration. (PDF 80 kb)

Supplementary Video 1

Anopheles gambiae embryo alignment for microinjection. (MOV 66430 kb)

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Pondeville, E., Puchot, N., Meredith, J. et al. Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae. Nat Protoc 9, 1698–1712 (2014). https://doi.org/10.1038/nprot.2014.117

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