standard φC31 in Anopheles gambiae: A How-To

Omar Akbari, Ph.D., California Institute of Technology

Omar Akbari, Ph.D., California Institute of Technology

In terms of genetic engineering of insects, the current most commonly used technique for germline transformation involves the use of mobile transposable genetic elements, which are simply inefficient, have limited carrying capacities, and integrate randomly into genomes resulting in insertional mutagenesis and position effects.

To circumvent many of these issues, an efficient site-specific integration system discovered in bacteria (Thorpe and Smith, 1998), known as the ΦC31 system, has been adapted to many organisms including several mosquito species such as the dengue vectors Aedes aegypti (Nimmo et al. 2006) and Aedes albopictus (Labbe et al. 2010), and the malaria vector Anopheles gambaie (Meredith et al. 2011). This system utilizes a sequence-specific recombinase ΦC31 that mediates recombination between two attachment sites (attP and attB), one positioned in the insect genome (attP), and the other in the plasmid DNA to be integrated (attB). A highly efficient recombination event between these two sequences results in the production of unique hybrid sites, (attL) and (attR), that are no longer recognized by the integrase, rendering insertions both unidirectional and stable. Essentially, this system gives scientists the ability to efficiently place desired pieces of DNA at specific locations in the genome consistently.

Simple phiC31-mediated site-specific integration of a transgene-containing Plasmid into a genome containing an appropriate attachment site (attP)

Simple phiC31-mediated site-specific integration of a transgene-containing Plasmid into a genome containing an appropriate attachment site (attP)

Pondeville et al. (2014) present an optimized step-by-step protocol, including a video, for efficient ΦC31-mediated site-specific integration of transgenes into the genome of the malaria vector Anopheles gambiae.

Briefly, the protocol begins with preparation of plasmid DNA harboring a desired attB-transgene for injection.  Once prepared, this plasmid DNA can be injected directly into self-docking strains that contain both attP integration sites and also express ΦC31 recombinase in their germline (Meredith et al. 2013).  Alternatively, when self-docking sites are not available, the plasmid DNA can be mixed with an exogenous source of ΦC31 recombinase that can either be expressed from a co-injected plasmid, or produced in vitro as mRNA, and then microinjected into embryos containing a genomic attP attachment site.  Once injected, the G0 embryos are hatched and outcrossed. The G1 progeny from this cross are screened for transgenesis using fluorescence, and transgenic individuals are then crossed to generate a stable homozygous line. This entire process from injection to homozygous line only takes ~2-3 months.

Anopheles gambiae feeding on a human.  photo credit: CDC

Anopheles gambiae feeding on a human. photo credit: CDC

To put this in perspective, it takes about the same amount of time to establish homozygous transgenic lines in in the fruit fly Drosophila melanogaster. Therefore, given the speed of genetics, a sequenced genome, and the rapid advancement of molecular tools including efficient transgenesis, the mosquito is becoming more of a model organism to work with in the laboratory. Furthermore, while this protocol has been optimized for Anopheles gambaie, it can straightforwardly be modified for other mosquito species.

Overall, this protocol presented by Pondeville et al. will likely help facilitate the development of genetics-based control methods for the mosquito, and is also an invaluable resource for basic research.  It provides many useful insights in to the details of injecting embryos of An. gambiae, a challenging species of mosquito work with in the laboratory.

This video is from Ponderville et al (2014)

Pondeville, E. et al. Efficient Phi C31 integrase-mediated site-specific germline transformation of Anopheles gambiae. Nat Protoc 9, 1698-1712, doi:Doi 10.1038/Nprot.2014.117 (2014).

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