Generation of transgenic anopheline mosquitoes refractory to malaria parasite by using gene manipulation

在与克里桑蒂教授的合作中，首席研究员克隆了冈比亚按蚊羧肽酶（AgCP）基因的启动子区域。制备两种质粒构建体（胞质和分泌形式），其编码在AgCP启动子控制下连接至Shiva的13.1单链抗体（13.1 scFv-Shiva）。将携带胞质基因的质粒DNA注射到斯氏按蚊胚胎中。经荧光阳性筛选，获得了5个转基因品系。13. 1 scFv-Shiva基因在转基因蚊子的肠道中大量表达。13.1 scFv-Shiva的表达是肠道特异性的，并在血液摄入后约3小时达到峰值水平。AgCP启动子可用于驱动阻碍蚊子肠道中寄生虫发育的基因的表达。为了测量13.1 scFv-Shiva转基因表达对寄生虫发育的影响，我们在同一感染小鼠上饲喂对照和转基因蚊子，并测量所形成的卵囊的数量。转基因蚊子中的卵囊形成与野生型蚊子中的卵囊形成不可区分。Crisanti教授的研究小组表明，蜂毒磷脂酶抑制转基因蚊子中疟原虫的发育（JBC 2002）。他们还表明，蚊子的基因改造为控制疟疾提供了令人兴奋的可能性，但成功与否将取决于传播如何影响改造昆虫的适应性（Science 2003）。Sinden教授的小组加入了啮齿动物疟原虫基因组序列的项目。序列信息提供了对疟原虫生物学和疾病的深入了解（Nature 2002）。因此，我们已经建立了产生转基因蚊子的技术和设施。这项工作代表了一个重要的一步，朝着发展的分子遗传学方法来控制疟疾传播的载体能力。

In collaboration with Prof.Crisanti, the head investigator cloned the promoter region of the Anopheles gambiae carboxypeptidase (AgCP) gene. Two plasmid constructs (cytoplasmic and secreted forms) encoding the 13.1 single-chain antibody linked to Shiva (13.1 scFv-Shiva) under the control of the AgCP promoter were made. The plasmid DNA harboring the cytoplasmic gene was injected into Anopheles stephensi embryos. Using fluorescent positive selection, 5 lines of transgenic mosquitoes were obtained. The 13.1 scFv-Shiva gene was abundantly expressed in the guts of transgenic mosquitoes. Expression of the 13.1 scFv-Shiva was gut-specific and reached peak levels at about 3 h post-blood ingestion. The AgCP promoter can be used to drive the expression of genes that hinder parasite development in the mosquito gut. To measure the consequences of the 13.1 scFv-Shiva transgene expression on the parasite development, we fed control and transgenic mosquitoes on the same infected mouse and measured the numbers of oocysts formed Oocyst formation in transgenic mosquitoes was indistinguishable from that in wild type mosquitoes. Another line expressing the secreted form of 13.1 scFv-Shiva is in progress of examining.Prof.Crisanti's group showed that bee venom phospholipsase inhibits malaria parasites development in transgenic mosquitoes (JBC 2002). They also showed that genetic modification of mosquitoes offers exciting possibility for controlling malaria, but success will depend on how transmission affects the fitness of modified insects (Science 2003).Prof.Sinden's group joined the projects on genomic sequence of rodent malaria parasites. The sequence information provides insight into Plasmodium biology and disease (Nature 2002).Thus, we have established technology and facility for generating transgenic mosquitoes. This work represents a significant step toward the development of molecular genetic approaches to the control of vector competence in malaria transmission.