Genetic Manipulation of Anopheles Mosquitoes

Anopheles gambiae mosquitoes (G3 and Ngousso strains and their transgenic derivatives) were maintained at 28°C and 70-80% humidity in a 12/12 h day/night cycle. Anesthetized CD1 mice were used for blood feeding and larvae were fed finely ground Tetra Goldfish food (Tetra, Germany). The DSX transgenic strain, obtained in the G3 background, has been previously described [18 (link)].
The FK transgenesis plasmid was assembled by recombining three entry plasmids and one piggyBac destination plasmid using three-fragment Multisite Gateway cloning (Invitrogen) according to the manufacturer’s instructions. The resulting construct contained the attB1, attB2, attB3 and attB4 Gateway seam sites that delimit each of the three cassettes contributed by the entry plasmids (for the full annotated construct sequence refer to Additional file 1). The Vitellogenin (AGAP004203) promoter was amplified from A. gambiae genomic DNA using the following primers: 5’-TGACCTCGAGTTCAACTCGACC-3’ and 5’-GATATCGATGGTTCGGTTGTTCGCAGTTG-3’. The amplified fragment was cloned into Xho I and Cla I restriction sites of the YFP-containing entry vector. The AGAP002620 promoter region was amplified using primers: 5-CCGTCTAGACCGGGCTCTACAAAGTC-3’ and 5’-CAGCTCTCGAGCAGGAGGATCGTT-3’ and cloned as an Xba I - Xho I fragment into the tdTomato-containing entry vector. Embryos (n = 120) of the Ngousso strain were injected with a 200 ng/μl solution of the transgenesis plasmid and 20 surviving adults were back-crossed to Ngousso mosquitoes. A single transgenic mosquito male was recovered from the back-cross progeny. Further genetic crosses revealed that the transgene insertion was X-linked. The piggyBac insertion was mapped by inverse PCR as follows: 500 ng of genomic DNA were digested with Sau3AI or cocktails of blunt end restriction enzymes (ScaI HincII, DraI, SmaI PvuII, Fermentas), and re-ligated with T4 DNA ligase (Fermentas) in a final volume of 500 μl. The sample was ethanol-precipitated, re-suspended in 20 μl water, of which 2 μl were subjected to PCR. The piggyBac 5’ border of the insertion site was mapped by sequencing a product amplified with primers 5’-TGCACAGCGACGGATTCGCGCTATT-3’and 5’-AGGACATCTCAGTCGCCGCTTGGA-3’, followed by nested PCR with 5’-CGCGCTATTTAGAAAGAGAGAG-3’ and 5’-GAACTATAACGACCGCGTGAGTC-3’; or with 5’-GAACTATAACGACCGCGTGAGTC-3’ and 5’-CAGTGACACTTACCGCATTGACA-3’. The piggyBac 3’ border of the insertion site was mapped by sequencing the product amplified with primers 5’-CGAGGTTTATTTATTAATTTGAATAGATATTAAG-3’ and 5’-CGATATACAGACCGATAAAACACATGCGT-3’, followed by nested PCR with 5’-GCGTCAATTTTACGCATGATTATCTTT-3’ and 5’-ATTTACACTTACATACTAATAATAAATTCAAC-3’.
Amplified fragments were compared by BLAST to the Anopheles gambiae genome (VectorBase). The transposon insertion was mapped within a 232-base pair repeated element on the X chromosome. This short repeated element is broadly distributed in the genome, but at the position X: 22463464 is present in the Ngousso strain and absent from the G3 and PEST strains. The transgenic construct carried the EGFP gene under the control of a 3xP3 promoter [19 (link)] as a transgenesis selection marker, and two additional reporter genes: (i) YFPvenus[20 (link)] under the control of the A. gambiae Vitellogenin (AGAP004203) promoter and (ii) tdTomato[21 (link)] under the control of the AGAP002620 gene promoter [22 (link)]. The detailed characterization of these additional reporter constructs will be described elsewhere. Mosquitoes were reared and blood-fed on anesthetized mice in compliance with French and European laws on animal house procedures (agreement #E67-482-2 of the Direction of Veterinary services of the French Ministry of Agriculture).