title: Efficient ΦC31 integrase-mediated site-specific germline transformation of Anopheles gambiae.
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.
authors.affiliation: Institut Pasteur, Genetics and Genomics of Insect Vectors, CNRS unit URA3012, Paris, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Pondeville
authors.affiliation: Institut Pasteur, Genetics and Genomics of Insect Vectors, CNRS unit URA3012, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Puchot
authors.affiliation: Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK.
authors.forename: Janet M
authors.initials: JM
authors.lastname: Meredith
authors.affiliation: Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Lynd
authors.affiliation: Institut Pasteur, Genetics and Genomics of Insect Vectors, CNRS unit URA3012, Paris, France.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Vernick
authors.affiliation: 1] Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK. [2].
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Lycett
authors.affiliation: 1] Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK. [2].
authors.forename: Paul
authors.initials: P
authors.lastname: Eggleston
authors.affiliation: 1] Institut Pasteur, Genetics and Genomics of Insect Vectors, CNRS unit URA3012, Paris, France. [2].
authors.forename: Catherine
authors.initials: C
authors.lastname: Bourgouin
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000852:Anopheles / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases / Q000235:genetics*; D007814:Larva / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D012333:RNA, Messenger; D019426:Integrases