Nanoject 2 auto nanoliter injector

To obtain X. laevis embryos, female frogs were induced to ovulate by injecting 500 to 700 U of human chorionic gonadotropin subcutaneously, directly above the dorsal lymph sacs. After injection, females were maintained in the dark at 18°C overnight. After females began ovulating the following morning, one male was euthanized by placing it in 0.1% tricaine solution buffered with sodium bicarbonate for 30 min. Both testes were dissected and placed into a 35-mm dish containing 1.5 ml of 1× Mark’s modified ringer (MMR) solution [0.1 M NaCl, 2 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, and 5 mM Hepes (pH 7.8)]. Eggs were gently squeezed from females into 10-cm plates. Sperm were released to fertilize the eggs by mincing a small portion of the testes in a few drops of 1× MMR placed beside the eggs followed by gentle mixing of eggs and sperm together. After 10 min at room temperature (RT), the fertilized eggs were flooded with 0.1× MMR solution (pH 7.4). Embryos were dejellied using 2% l-cysteine solution (pH 7.8) for 2 min. Embryos were washed thrice with 0.1× MMR solution and placed in 0.1× MMR. At the desired developmental stage, embryos were injected with a Nanoject II Auto-Nanoliter Injector (Drummond Scientific Inc.). Injected embryos were maintained in 4% Ficoll/0.1% MMR solution at 18°C until they reached stage 9 and then transferred to 0.1× MMR buffer.

RNA interference was performed as previously described (Povelones et al., 2011 (link); Schleicher et al., 2018 (link)). dsRNA targeting mosGILT, TEP1, CTL4, or an irrelevant luc gene from Renilla reniformis (Schleicher et al., 2018 (link)), were transcribed using the MEGAScript RNAi kit (Thermo Fisher Scientific, Ambion; primers in Table S1).
2-d-old A. gambiae mosquitoes were injected with dsRNA using a Nanoject II Auto-Nanoliter Injector (Drummond) and allowed to recover in paper cups with 10% sucrose for 2 d. For silencing of CTL4 or TEP1, 0.35 µg of total dsRNA was used for each mosquito. For silencing of mosGILT, 1.0 µg of total dsRNA was injected into each mosquito. On day 4 after dsRNA injection, these mosquitoes were deprived of sucrose for 20 h before allowing them to feed on P. berghei–infected mice with 3–5% parasitemia. Unfed mosquitoes were immediately discarded, and the fed remaining mosquitoes were maintained with 10% sucrose containing 0.05% penicillin/streptomycin for 8 d at 20°C. On day 8 after infection, oocysts and melanized ookinetes in the midgut were counted as mentioned above.