Construction of transgenic mosquitoes for foreign gene expression

利用基因工程技术，对日本产抗病原菌蚊子的生产工艺进行了研究。日本蚊子基因组中水手样基因的存在。利用mariner样基因的反末端重复序列（ITR）作为引物扩增mariner总长度。结果表明，ITR基因在白纹伊蚊和按蚊中均有扩增。然而，当以mariner基因编码转移酶的保守区域作为引物时，仅在白纹伊蚊中观察到PCR产物。由此推测，白纹伊蚊的水手样基因可能是其中一个活跃的转座子。蚊卵注射后的孵化率。将转座子hobo基因和辅助转座酶的混合dna注射到发育90分钟后的埃及伊蚊卵中。之后，观察到它们的孵化率更高。结果，39%的埃及伊蚊卵被注射后孵化为正常幼虫。然而，注射DNA溶液的白纹伊蚊卵没有孵化。结果表明，不同蚊种对蚊卵的注射条件不同。蚊卵电穿孔后的孵化率。作为将mariner [acti5c - egfp]基因插入蚊子基因组的方法，研究了注射法和电穿孔法。使用Mariner [actic - egfp] DNA作为人工转座子和辅助（Mariner） DNA混合物。脉冲装置电穿孔法孵化埃及伊蚊卵。但未见白纹伊蚊卵孵化。提示不同蚊种的适宜脉冲条件不同。构建了以日本蚊活性转座子为载体的人工转移载体，在日本蚊活性转座子mariner元件上游构建了增强子，在杆状病毒中构建了ie1启动子，在下游构建了GFP基因作为标记。本报告描述了更多细节。少

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