Femtojet equipment

Aedes aegypti Liverpool strain (LVP) was used for all experiments and were kept confined in chambers in an insectary at 28 °C, 60–70% humidity and 14:10 h light:dark cycle. The colony was maintained, and experiments were performed exclusively on defibrinated sheep blood (Colorado Serum Company, Denver, CO, USA) using an artificial feeding system. The generation of Ae. aegypti transgenic lines was performed as described previously [26 (link)]. Briefly, preblastoderm embryos were microinjected with a mixture of one of the donor plasmids (bZip-mNG, bZip-tTA or bZip-tTA-mNG) at 0.5 μg/μl and the helper plasmid (pGL3-pUbMos) [31 (link), 32 (link)] at 0.3 μg/μl using FemtoJet® equipment (Eppendorf, Hamburg, Germany) and pulled borosilicate glass capillaries (World Precision Instruments, Sarasota, USA). Females from surviving injected embryos were pooled into 20–25 individuals per cage and backcrossed to LVP in a 1:1 ratio, while males were mated with LVP females in a 1:5–10 ratio. The G₁ progeny from injected embryos were screened for DsRed of the marker gene during larval stages. Transgenic lines obtained from female and male pools were identified by the letter P and F, respectively, followed by a number representing the cage they came from (e.g. bZip-mNG P3 or bZip-mNG F1). All experiments were performed with heterozygous mosquitoes.

Ae. aegypti Liverpool strain (LVP) were maintained at 28°C, 60–70% humidity and 14h light/ 10h dark cycle in chambers in an insectary. Blood feeding was performed exclusively on defibrinated sheep blood (Colorado Serum Company, Denver CO, USA) using an artificial feeding system for maintenance. The generation of Ae. aegypti transgenic lines was performed as described previously [19 (link)]. Briefly, preblastoderm embryos were microinjected with a mixture of pTRE-Nix/NixPro-tTA donor plasmid at 0.5μg/μL and the helper plasmid (pHSP-Bac) [20 (link)] at 0.3μg/μL using FemtoJet equipment (Eppendorf, Hamburg,Germany) and pulled borosilicate glass capillaries (World Precision Instruments, Sarasota, USA). Females from surviving injected embryos were pooled into ~20 individuals per cage and backcrossed to LVP in a 1:1 ratio, while males were mated with LVP females in a 1:5–10 ratio. The G₁ progeny from injected embryos were screened for DsRed during larval stages. Transgenic lines obtained from female and male pools were identified by the letter P and F respectively, followed by a number representing the cage they came from (e. g. P5 or F3). All experiments were performed with transgenic heterozygous mosquitoes.