1. Efficient PhiC31 integrase-mediated site-specific germline transformation of Anopheles gambiae
- Author
-
Kenneth D. Vernick, Nicolas Puchot, Catherine Bourgouin, Gareth J Lycett, Emilie Pondeville, Amy Lynd, J. M. Meredith, Paul Eggleston, Génétique et Génomique des Insectes vecteurs, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Keele University [Keele], Liverpool School of Tropical Medicine (LSTM), 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. 223736 (TransMalariaBloc) and a Biotechnology and Biological Sciences Research Council grant (no. BB/F021933/1)., 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., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 323173,EC:FP7:ERC,ERC-2012-ADG_20120314,ANOPATH(2013), European Project: 228421,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2008-1,INFRAVEC(2009), European Project: 223736,EC:FP7:HEALTH,FP7-HEALTH-2007-B,TRANSMALARIABLOC(2008), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Liverpool School of Tropical Medicine ( LSTM ), ANR-10-LABX-62-IBEID,IBEID,Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' ( 2010 ), European Project : 323173,EC:FP7:ERC,ERC-2012-ADG_20120314,ANOPATH ( 2013 ), European Project : 228421,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2008-1,INFRAVEC ( 2009 ), European Project : 223736,EC:FP7:HEALTH,FP7-HEALTH-2007-B,TRANSMALARIABLOC ( 2008 ), and Centre National de la Recherche Scientifique ( CNRS ) -Institut Pasteur [Paris]
- Subjects
MESH : RNA, Messenger ,MESH: Integrases ,[ SDV.BA.ZI ] Life Sciences [q-bio]/Animal biology/Invertebrate Zoology ,Anopheles gambiae ,MESH : Animals, Genetically Modified ,Messenger/metabolism ,Genome ,Germline ,Animals, Genetically Modified ,Genetic Engineering/*methods ,Plasmid ,MESH: Animals ,Genetics ,[ SDV.SPEE ] Life Sciences [q-bio]/Santé publique et épidémiologie ,3. Good health ,Genetically modified organism ,Transformation ,MESH : Anopheles ,Larva ,MESH: Genetic Engineering ,Genetic Engineering ,Functional genomics ,[ SDV.MP.PAR ] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,Transgene ,Genetically Modified ,MESH: Transformation, Genetic ,Biology ,Integrases/*genetics ,General Biochemistry, Genetics and Molecular Biology ,MESH : Genetic Engineering ,MESH: Animals, Genetically Modified ,MESH: Anopheles ,MESH : Integrases ,Transformation, Genetic ,Genetic ,Anopheles ,parasitic diseases ,Animals ,[SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology ,RNA, Messenger ,Anopheles/*genetics ,MESH: RNA, Messenger ,MESH : Larva ,Integrases ,fungi ,Larva/genetics ,biology.organism_classification ,[SDV.BA.ZI]Life Sciences [q-bio]/Animal biology/Invertebrate Zoology ,Transformation (genetics) ,RNA ,[SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie ,MESH : Animals ,MESH : Transformation, Genetic ,MESH: Larva - Abstract
International audience; 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 PhiC31-mediated site-specific integration of transgenes into the genome of A. gambiae. The PhiC31 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 PhiC31 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 approximately 2-3 months.
- Published
- 2014
- Full Text
- View/download PDF