Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Adolfi, Adriana, Lynd, Amy ORCID: https://orcid.org/0000-0001-6054-0525, Lycett, Gareth ORCID: https://orcid.org/0000-0002-2422-053X and James, Anthony A. (2021) 'Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria'. Journal of Visualized Experiments (JoVE), Vol 168, e62146.

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Abstract

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

Item Type:	Article
Subjects:	QU Biochemistry > Genetics > QU 460 Genomics. Proteomics QX Parasitology > Insects. Other Parasites > QX 515 Anopheles WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department:	Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI):	https://doi.org/10.3791/62146
Depositing User:	Mel Finley
Date Deposited:	05 Mar 2021 15:20
Last Modified:	05 Mar 2021 15:20
URI:	https://archive.lstmed.ac.uk/id/eprint/17162

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