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

This protocol supports the characterization of the role of mosquito genes involved in a variety of physiological pathways, including, for example, insect resistance and malaria parasite development. The method drives transgene insertion into single pre-characterized genomic locations and therefore allows to directly compare phenotypes resulting from alternate transgenes effectively avoiding the issue of positional effect. This method can be applied to a variety of insect species of public health and agricultural importance and its applications extend widely from bacteria to mammalian cells.

Begin by designing attB donor plasmids carrying the dominant fluorescent marker, attB recombination sites and the desired transgene cargo. Use a single attB site for integration of the whole plasmid carrying the transgenic cassette or two inverted attB sites for cassette exchange. Purify donor and helper plasmids using an endotoxin-free purification kit.

Combine the attB tagged donor plasmid carrying the transgene of interest and the helper plasmid carrying the integrase to obtain a mix with a final concentration of 350 nanograms per microliter of the donor plasmid and 150 nanograms per microliter of the helper plasmid. Precipitate the DNA by adding 0.1 volume of three molar sodium acetate and 2.5 volumes of ice cold 100%ethanol, then vortex. A white precipitate should be immediately visible.

Wash the pellet and resuspend it in injection buffer to reach a total final concentration of 500 nanograms per microliter. Then prepare aliquots of 10-15 microliters each and store them at negative 20 degrees Celsius. Blood feed four to seven-day-old mosquitoes from the desired docking line and their wild type counterparts 72 hours prior to micro injection.

Perform Anopheles gambiae embryo micro injections in 25 millimolar sodium chloride by targeting the posterior pole of the embryo at a 45 degree angle. Perform Anopheles stephensi embryo micro injections in halocarbon oil by targeting the posterior pole at a 30 degree angle. Immediately after injection, transfer the eggs to a Petri dish filled with sterile distilled water and return them to insectary conditions.

Upon hatching, transfer G0 larva into a tray with salted distilled water daily and rear to pupae. Sort G0 pupae by sex under a stereoscope. Allow the males to emerge in separate cages in groups of three to five and add a tenfold excess of age-matched wild type females.

Allow the females to emerge in separate cages in groups of 10 to 15 and add an equal number of age-matched wild type males. Allow adults to mate for four to five days and provide females with a blood meal. Collect the eggs and rear emerging next generation G1s.

Collect G1, L3, and L4 larva in a Petri dish lined with wet filter paper or on a microscope slide and screen them using a fluorescent stereoscope for the presence of the marker introduced with the attB tagged cargo. For single attB designs, screen for the presence of the new and preexisting marker. For double attB designs for cassette exchange, screen for the presence of the new marker and the loss of the preexisting one.

Sort transformed G1 pupae by sex and cross them en masse with opposite sex age-matched wild type individuals. Allow adults to mate for four to five days and provide a blood meal. For single integration experiments, collect eggs directly from the en masse cross.

For cassette exchange experiments, collect eggs from single females and maintain progeny separate until molecular assessment is complete due to the potential presence of two alternative cassette orientations. Screen the G2 progeny for the presence of the fluorescent marker and set aside a subset of G2 positive individuals for molecular analysis. Rear the rest to adulthood.

Perform molecular validation of the insertion sites with PCR as described in the text manuscript. For single integration, ensure that the predicted insertion site carries the original docking construct, plus the whole sequence of the donor plasmid between the two hybrid sites attL and attR. For cassette exchange, ensure that the predicted insertion site is identical to that of the docking line where hybrid inverted attL sites replace the original inverted attP sites and the exchange template replaces the cassette originally present between them.

The protocol was used to generate a stable Anopheles transgenic line in approximately 10 weeks. Phenotypic validation of transformation was performed by screening for fluorescent markers regulated by the 3xP3 promoter are shown here. A new Anopheles stephensi line was obtained by insertion of a DS red marked cassette into a docking line marked with CFP which resulted in G1 progeny expressing both markers as indicated by the red and blue fluorescence in the eyes.

Cassette exchange designs result in the replacement of the marker originally inserted into the docking line with that of the donor plasmid. This marker exchange was demonstrated in an Anopheles gambiae docking line where the CFP marker was lost and the YFP marker acquired resulting in yellow eye and nerve cord fluorescence. Occasionally, RMCE can result in a single integration event instead of the exchange of the desired transgenic cassette where a larva is marked with both the original CFP and the new YFP markers.

When screening for the presence of a fluorescent marker, it is crucial to distinguish its signal from possible background auto-fluorescence. Increasing the magnification and focusing on the tissues and organs where fluorescence is expected to be driven by the promoter is necessary to identify true CFP positive individuals. Individual transformants were also assessed molecularly via PCR to confirm the expected insertion site.

PCR validation in individuals from an exchange Anopheles gambiae line is shown here. Even appropriate micro injection technique, the accurate design and preparation of the donor and helper plasmids, as well as following the appropriate mosquito husbandry scheme are key for obtaining transgenic individuals. This procedure can be used to insert elements causing gene over-expression or silencing, for example, the GAL4/UAS system, as well as gene drive elements and anti-pathogen molecules for mosquito genetic control.