Construction of transgenic mosquitoes for foreign gene expression
Grant-in-Aid for Scientific Research (C)
|Allocation Type||Single-year Grants |
Laboratory animal science
|Research Institution||OITA MEDICAL UNIVERSITY |
ESHITA Yuki Oita Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10082223)
FUKUDA Masako Oita Medical University, Faculty of Medicine, Research Associate, 医学部, 助手 (00156788)
MATSUMOTO Akira Kyushu University Research Center for Higher Education, Research Associate, 大学教育研究センター, 助手 (40229539)
|Project Period (FY)
1999 – 2001
Completed (Fiscal Year 2001)
|Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
|Keywords||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 / 反復逆配列 / 外来遺伝子の発現|
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
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 mosquitoes
Enhancer 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
Report (4 results)
Research Products (27 results)