LA OBRA DEL TEMPLO NO ES PARA LOS REBELDES

2.4.2.1 Mosquito lines

Details of mosquito maintenance are reported in Appendix A. In this study, six mosquito lines were used:

- G3: wild type strain, originally isolated from The Gambia, obtained from the Malaria Research and Reference Reagent Resource Centre (MR4).

- A11: transgenic homozygous docking line created by piggyBac-mediated integration. The transgene cassette is inserted in 2R: 33,858,877-80 (Lynd et al.,

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unpublished) and is flanked by attP sites for PhiC31-RMCE. The line is marked with 3xP3-driven cyan fluorescent protein (CFP).

- GAL4mid: transgenic homozygous driver line created by piggyBac-mediated integration. It expresses GAL4 under the control of the An. gambiae carboxypeptidase promoter (Moreira et al., 2000) which directs expression in the adult midgut. The transgene cassette lays in an imprecisely mapped site on the 3R chromosome (Lynd & Lycett, 2012) and the line is marked with 3xP3-driven Discosoma sp. red fluorescent protein (DsRed).

- GAL4oeno: transgenic homozygous driver line created by piggyBac-mediated enhancer trapping. It directs expression of GAL4 in the oenocytes of all life stages. The transgene cassette is inserted in 2R: 48,392,077-80 (Lynd et al., unpublished) and line is marked with 3xP3-driven DsRed.

- UASm2 and UASp3: transgenic responder lines created by PhiC31-RMCE using the A11 docking strain. They are marked with 3xP3-driven YFP and designed for the expression of Cyp6m2 and Cyp6p3 respectively. Details on the creation of these lines are reported in sections 2.4.2.3 and 2.5.1.

2.4.2.2 Microinjections

Transgenic mosquitoes were produced by site-specific germline transformation (Pondeville et al., 2014) using PhiC31-RMCE.

2.4.2.2.1 DNA and needle preparation for microinjection

To prepare the pUASm2 injection mix, plasmid DNAs were harvested from bacterial cultures using Plasmid Midi Kit (Qiagen), ethanol precipitated and resuspended to obtain a solution containing 350 ng/µl of the pUASm2 responder plasmid and 150 ng/µl of the integrase helper plasmid PKC40 (Ringrose, 2009) in a total volume of 20 µl of 1x injection buffer (5 mM KCl, 0.5 mM NaPO4, pH 7.2) (Lombardo et al., 2009).

For pUASp3, an injection solution with a final concentration of 330 ng/µl was prepared instead at the same ratio of donor to helper plasmids. These mixes were used in two independent microinjection experiments.

Injecting needles were pulled from quartz capillaries with filament (fire polished) (Sutter Instrument Co.) with a length of 7.5 cm, 1 mm outer diameter and an inner diameter of 0.7 mm. Needles were prepared using Sutter Instrument Co. P-2000

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Micropipette Puller with the following setting: HEAT = 650; FIL = 4; VEL = 25; DEL = 145; PUL = 200.

2.4.2.2.2 Embryo alignment and microinjection

Adult female mosquitoes of the A11 docking strain were allowed to lay in water for 20 minutes in the dark and embryos left to develop for further 30 minutes in insectary conditions.

Eggs were transferred in 25 mM NaCl onto a microscope slide and arranged in lines against the edge of Whatman filter paper grade 1 (Sigma). All embryos were aligned so the posterior (thinner) pole was exposed for injection and, whenever possible, so they laid on their dorsal side. After completing a line of ~50 embryos, Whatman paper was dried and removed, and eggs transferred onto a cover slip with double sticky tape. Here they were left to dry for 30 seconds before recovering them in a drop of 25 mM NaCl. Alignments were conducted using Leica MZ FLIII stereoscope at room temperature.

Microinjections were carried out in 25 mM NaCl at 200x magnification using Nikon Eclipse TE2000-U inverted microscope and Eppendorf® _FemtoJet® _{Microinjector (Pi}

= 2000-3000 HPa) at room temperature (Lombardo et al., 2009). After injection, eggs were immersed in 2-3 cm of mineral water and returned to insectary conditions.

2.4.2.3 Creation of the UASm2 and UASp3 responder lines

Emerging F0 larvae were separated by sex at pupal stage and surviving F0 adults

were pooled into sex specific founder cages and outcrossed with 3x excess of wild type G3s of the opposite sex. Five days after crossing, a first blood meal was offered and larvae of the F1 progeny screened for YFP expression only (cassette exchange),

CFP expression only (no exchange) or CFP/YFP expression (cassette integration) in the eyes and nerve cord.

For the UASm2 line, a second blood meal was offered to the founder cages and 16 individual female lays were set up to obtain F1 isofemale lines. To do so, universal 30

ml plastic vials containing wet cotton wool and Whatman filter paper grade 3 (Sigma) were prepared and, two days after blood-meal, single females transferred inside and left laying overnight. Papers containing eggs were transferred into individual trays to

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hatch. Females that did not lay were left in the vials for another night, after which they were discarded if did not lay eggs.

For the UASp3 lines, 19 isofemale lines were set up as described above from F1

transgenic females after crossing with G3s. Orientation of insertion was then assessed in corresponding parental females and a single isofemale line established as the definitive line either by crossing transformant individuals with G3s (UASm2) or by interbreeding them with negative adults of the same batch (UASp3). Responder lines were kept as a mix of homozygous and heterozygous individuals so to obtain GAL4/+ progeny to be used as transgenic blank controls.

Minimum transformation efficiency was calculated as (Number of independent insertions / total F0 adult survivors) x 100.

2.4.2.3.1 Orientation of insertion

Cassette exchange may happen in two different orientations with respect to the chromosome, designated A or B (Figure 2.3). Orientation check was performed on the parental females and on the controls G3 and A11 by PCR. Combinations of 4 primers designed to give a product only in one of the orientations were used in 4 different PCRs: piggyBacR-R2 + Red-seq4R for PCR 1, M2intFW or P3intFW + ITRL1R for PCR 2, piggyBacR-R2 + M2intFW or P3intFW for PCR3, and Red-seq4R + ITRL1R for PCR 4 (Figure 2.3). PCRs were performed using Phire Hot Start II DNA Polymerase (Life Technologies) and a 30-cycle thermal program consisting of 60°C annealing temperature and 60-second extension.

Definitive UASm2 and UASp3 isofemale lines were created from individuals showing orientation of insertion A, which was chosen consistent with previous RMCE lines created in this laboratory.

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Figure 2.3 Two possible orientations of insertion, A and B, after recombination of the attP sites in the docking mosquito genome and the attB sites in the UAS-Cyp6 plasmids. attP, attB: PhiC31 recombination sites; CFP: cyan fluorescent protein; YFP: yellow fluorescent protein; UAS: upstream activating sequence; attL, attR: hybrid sites created after recombination; →: primer annealing site, 1: piggyBacR-R2; 2: Red-seq4R; 3: M2intFW or P3intFW; 4: ITRL1R (Appendix B, Table B.1).