phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector

1. Preparation of plasmids for the microinjection mix

NOTE: The protocol illustrated here involves the use of two plasmids: an attB (attachment site derived from the host bacterium)-tagged donor plasmid carrying the transgene of interest, and a helper plasmid that expresses the φC31 integrase under the regulation of the Drosophila Hsp70 promoter.

1. Purify donor and helper plasmids using an endotoxin-free plasmid purification kit.
   NOTE: Sequence the final plasmid preparation used for injection to verify the integrity of all components.
2. Combine appropriate amounts of the two plasmids to obtain a mix with a final concentration of 350 ng/µL of the donor plasmid and 150 ng/µL of the helper plasmid when resuspended in injection buffer.  
   NOTE: When calculating the necessary volume of mix, consider that 10-15 µL are sufficient for each day of planned injections and DNA can be prepared in advance and stored at -20 °C. Integrase helper plasmid concentrations of 60-500 ng/µL and donor plasmid concentrations of 85-200 ng/µL have also been reported.
3. Precipitate the DNA by adding 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ice-cold 100% EtOH and vortex. A white precipitate should be immediately visible. Having highly concentrated initial plasmid preparations (i.e., ~1 µg/µL) improves precipitation efficiency.    
   NOTE: Stopping point – The precipitate can be stored at -20 °C overnight.
4. Centrifuge at 15,000 x g for 20 min at 4 °C, discard the supernatant, and wash the pellet with 1 mL of ice-cold 70% EtOH.
5. Wash the pellet with 1 mL of ice-cold 70% EtOH and centrifuge at 15,000 x g for 5 min at room temperature.
6. Discard the supernatant without disturbing the pellet and air dry.
7. Resuspend the pellet in 1x injection buffer (0.1 mM Na₃PO₄, 5 mM KCl, pH 7.2, 0.22 µm filter sterilized) to reach a total final concentration of 500 ng/µL.
   NOTE: Assume that some DNA will be lost during the precipitation process; therefore, add a smaller volume of injection buffer first, check the concentration on a spectrophotometer (e.g., Nanodrop), and then add an appropriate remaining volume to reach 500 ng/µL.
8. Ensure that the DNA is thoroughly resuspended, prepare aliquots of 10-15 µL each and store them at -20 °C.
9. On the day of injection, thaw one aliquot and centrifuge at 15,000 x g for 5 min to remove any particulate residues.        
   ​NOTE: An alternative method for particulate removal is to filter the solution through a 0.22 µm filter. Avoid the presence of particulate residues in the injection mix as they lead to needle blockage during embryo microinjection.

2. Microinjection of embryos from an Anopheles docking line

1. Blood feed 4-7-day-old mosquitoes from the desired docking line 72 h prior to microinjection (i.e., for injection on Monday and Tuesday feed females on the previous Friday; for injection on Thursday and Friday feed females on Monday of the same week).
2. Blood feed wild-type (WT) mosquitoes (i.e., mosquitoes with the same genomic background of the docking line) on the same day; these will be needed for outcrossing.
   NOTE: The size and quality of the blood meal affect egg quality, so it is recommended to always use fresh blood (i.e., blood drawn within the previous 7 days). Arm feeding or feeding on mice may increase the quality and quantity of eggs, however these methods are not encouraged. Specific approved protocols will be necessary for human and animal use.
3. Perform embryo microinjections
   1. Perform An. gambiae embryo microinjections in 25 mM NaCl by targeting the posterior pole of the embryo at a 45-degree angle. For a detailed protocol for embryo collection, alignment, and microinjection refer to Pondeville et al. and Lobo et al.
   2. Perform An. stephensi embryo microinjections in halocarbon oil 700:27 (2:1) by targeting the posterior pole of the embryo at a 30-degree angle. A detailed protocol for embryo collection, alignment, and microinjection can be found in Terenius et al. and Lobo et al.
   3. Transfer eggs immediately after injection in a Petri dish filled with sterile distilled water (pH 7.2) and return them to insectary conditions.
4. Upon hatching, transfer G₀ larvae into a tray with salted distilled water (0.1% tonic salt) daily and rear to pupae.
5. Record hatching rate (i.e., number of larvae hatched/number of embryos injected).
   NOTE: Embryo movement aids hatching, so gentle swirling is desirable. Hatching should start ~48 h after injection. Since injection may cause a slight developmental delay, it is advisable to keep monitoring for late-hatching larvae for 3-4 days.