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

通过基因操作产生对疟原虫具有抵抗力的转基因按蚊

基本信息

批准号：
13670253
负责人：
YOSHIDA Shigeto
金额：
$ 1.92万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

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.

在Crisanti教授的合作下，首席研究员克隆了冈比亚按蚊羧基肽酶(AgCP)基因的启动子区域。在AgCP启动子的控制下，构建了与Shiva(13.1 scFv-Shiva)相连的13.1单链抗体的胞内型和分泌型表达载体。将含有胞质基因的DNA注射到斯氏按蚊胚胎中。通过荧光阳性选择，获得了5株转基因蚊虫。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，在摄血后约3小时达到峰值。AgCP启动子可以用来驱动阻碍蚊子肠道内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，我们用同一感染小鼠喂养对照和转基因蚊子，并测量了转基因蚊子形成卵囊的数量与野生型蚊子的卵囊形成没有区别。另一个表达13.1scFv-Shiva分泌型的品系正在检测中。Crisanti教授的研究小组表明，蜂毒磷脂酶在转基因蚊子中抑制疟疾寄生虫的发展(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良昆虫的适合性(Science 2003)。辛登教授的团队加入了啮齿动物疟疾寄生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洞察(自然2002)。因此，我们建立了生产转基因蚊子的技术和设施。这项工作代表着朝着开发分子遗传学方法控制疟疾传播中的媒介能力迈出了重要的一步。