Construction of transgenic mosquitoes for foreign gene expression

构建用于外源基因表达的转基因蚊子

• 批准号: 11680827
• 负责人: ESHITA Yuki
• 金额: $ 2.37万
• 依托单位: OITA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680827/
• 关键词: pathogen resistant mosquitoes; mariner genome; injection and electroporation methods; IE 1 promoter; GFP; seamless cloning method; SV40; baculovirus; 日本産蚊; mariner; transposase; 転写終結シグナル; actin5C; EGFP; トランスポゾン; 組換え蚊; 電気窄孔法; 注入法; ホーボ遺伝子

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

利用基因工程技术，研究了日本生产抗病原菌蚊子的程序。日本蚊虫基因组中存在水手样基因。以水手样基因的反向末端重复序列(ITR)为引物，扩增水手全长。结果表明，不仅在白纹伊蚊中存在ITR基因的扩增，而且在按蚊中也存在ITR基因的扩增。然而，以Mariner基因编码的转移酶保守区为引物时，仅在白纹伊蚊中观察到该产物。从这些观察结果可以推测，在白纹伊蚊中，Mariner-like基因作为一种活跃的转座子发挥作用。将转座子hobo基因和辅助转座酶基因的混合DNA注射到埃及伊蚊的卵中，实验进行了90分钟。之后，他们的…观察到更高的孵化率。结果表明，39%的埃及伊蚊幼虫作为正常幼虫孵化。然而，注射DNA溶液的白纹伊蚊卵未孵化，表明蚊卵的注射条件因蚊种而异。电穿孔后蚊卵的孵化率。作为一种将Mariner[actin5C-EGFP]基因插入蚊子基因组的方法，对注射和电穿孔两种方法进行了比较。使用Mariner[actin5C-EGFP]DNA作为人工转座子和辅助(Mariner)DNA的混合物。用脉冲仪电穿孔法孵化埃及伊蚊卵。但未观察到白纹伊蚊孵化卵。提示不同蚊种的适宜脉象不同。构建了日本蚊源活性转座子的人工转移载体，构建了日本蚊源活性转座子上游的增强子和杆状病毒来源的IE 1启动子的人工转移载体，下游以GFP基因为标记。本报告描述了更多细节。较少

项目成果

Uchida, K., Ohmori, D., Ueno, T., Nishizuka, M., Eshita Y., et al.: "Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens"Developmental Biology. 237. 68-78 (2001)

Uchida, K.、Ohmori, D.、Ueno, T.、Nishizuka, M.、Eshita Y.等人：“淡色库蚊退化卵泡中组织蛋白酶样蛋白酶的产前激活”发育生物学。

Uchida, K., Oda T., Matsuoka, H., Moribayashi, A., Ohmori, D., Eshita Y.: "Induction of oogenesis in mosquitoes (Diptera : Culicidae) by Infusion of the hemocoel with amino acids"J.Med.Entomol.. 38・4. 572-575 (2001)

Uchida, K.、Oda T.、Matsuoka, H.、Moribayashi, A.、Ohmori, D.、Eshita Y.：“通过向血腔注入氨基酸诱导蚊子（双翅目：蚊科）卵子发生”J。昆虫医学杂志.38・4.572-575 (2001)

Uchida, K., Ohmori, D., Ueuo, T., Nishizuka, M., Eshita Y., et al.: "Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex piplens pallens"Developmental Biology. 237. 68-78 (2001)

Uchida, K.、Ohmori, D.、Ueuo, T.、Nishizuka, M.、Eshita Y.等人：“淡色库蚊退化卵泡中组织蛋白酶样蛋白酶的产前激活”发育生物学。

Oda, T., Eshita, Y., Uchida, K., Mine, M., Kurokawa, K., et al.: "Reproductive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperature"J.Med.Entomol.. 39. 185-190 (2002)

Oda, T.、Eshita, Y.、Uchida, K.、Mine, M.、Kurokawa, K. 等人：“日本淡色库蚊和致倦库蚊（双翅目：库蚊科）的繁殖活动和生存状况

Morales, R., Morita, K., Eshita, Y., Tsuda, Y., Fukuma, T., Takagi, M.: "Infection and dissemination of two dengue type 2 viruses isolated from patients exhibiting different disease seventies in orally infected Aedes aegypti from different geographic orig

Morales, R.、Morita, K.、Eshita, Y.、Tsuda, Y.、Fukuma, T.、Takagi, M.：“从七十年代表现出不同疾病的口腔感染患者中分离出两种登革热 2 型病毒的感染和传播