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

Pondeville, E., Puchot, N., Meredith, J. M., Lynd, A., Vernick, K. D., Lycett, G. J., Eggleston, P. and Bourgouin, C. (2014) Efficient ΦC31 integrase–mediated site-specific germline transformation of Anopheles gambiae. Nature Protocols, 9(7), pp. 1698-1712. (doi: 10.1038/nprot.2014.117)

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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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Pondeville, Dr Emilie
Authors: Pondeville, E., Puchot, N., Meredith, J. M., Lynd, A., Vernick, K. D., Lycett, G. J., Eggleston, P., and Bourgouin, C.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Centre for Virus Research
Journal Name:Nature Protocols
Publisher:Nature Publishing Group
ISSN:1754-2189
ISSN (Online):1750-2799

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