Construction of transgenic mosquitoes for foreign gene expression

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Using the genetic engineering techniques, the procedure for producing the pathogen-resistant mosquitoes in Japan was examined.1. Existence of the mariner-like gene in Japanese mosquito genomes.Inversed terminal repeat (ITR) of the mariner-like genes as a primer was used for the amplification of the mariner overall length. As the results, there was the amplification of the ITR gene in not only Aedes albopictus mosquitoes but also Anopheles species. However, the PCR product was observed only in the Aedes albopictus mosquitoes, when conserved region in the transferase enzyme coded in the mariner gene was used as a primer. From these observations, it may be suggested that mariner-like gene functions in the Aedes albopictus mosquito as one of active transposons.2. Hatchability of mosquito eggs post injection.Mixture DNAs of the hobo gene that is a kind of the transposon, and of transposase as the helper were injected into eggs of Aedes aegypti post 90 minutes progress. And afterwards, their … More hatchability were observed. As the results, 39 % of Aedes aegypti eggs injected hatched as normal larvae. However, no Aedes albopictus eggs injected with DNA solutions hatched.It was indicated that injection condition to the mosquito egg differed by mosquito species.3. Hatchability of mosquito eggs post electroporation.As a method for inserting mariner [actin5C-EGFP] gene into mosquito genomes, injection and electroporation methods were examined. Mariner [actin5C-EGFP] DNA as an artificial transposon and helper (mariner) DNA mixture were used. Aedes aegypti eggs hatched by electroporation method using pulsed apparatus. However, no hatched egg of Aedes albopictus was observed. It was suggested that the suitable pulse condition differed in the mosquito species.4. Construction of an artificial transfer vector using an active transposon derived from Japanese mosquitoesEnhancer and IE 1 promoter derived from baculoviruses in the upperstream of an active transposon mariner element of Japanese mosquitoes, and the GFP gene as a marker in the downstream were constructed. More details were described in this report. Less