黄色猩々蝿のTALEモジュレーションを利用した転写因子群の二機能性に関する研究

2018 Fiscal Year Annual Research Report

• Project/Area Number: 18F18792
• Research Institution: Okinawa Institute of Science and Technology Graduate University
• Principal Investigator: ラスカム ニコラス 沖縄科学技術大学院大学, ゲノム・遺伝子制御システム科学ユニット, 教授 (70724087)
• Co-Investigator(Kenkyū-buntansha): CHEN ZHE 沖縄科学技術大学院大学, ゲノム・遺伝子制御システム科学ユニット, 外国人特別研究員
• Project Period (FY): 2018-10-12 – 2020-03-31
• Keywords: GENE REGULATION / STATISTICAL MODELLING / SYSTEMS BIOLOGY / DROSOPHILA MELANOGASTER

Outline of Annual Research Achievements

In FY2018, the fellow worked to establish a quantitative in situ hybridization protocol using the Gal4::rhoNEE-lacZ enhancer reporter and UAS::TALE::transcriptional activator in fruitfly embryos. The presence of the fluorophore, which was tagged to the enhancer reporter, was be observed by confocal microscopy, Zeiss LSM780, at OIST. Confocal microscopic images were acquired and fluorescent intensities proportional to the lacZ expression recorded.

Current Status of Research Progress

4: Progress in research has been delayed.

Reason

平成30年12月、本研究に使用する遺伝子組換生物作成の手法を確立するための事前準備過程で、参考としていた論文に記述されている方法ではプラスミド精製ができないことが判明した。そのため文献調査を再度行う必要が生じ、手法を確立するための事前準備期間を4カ月延長することとなった。

Strategy for Future Research Activity

Making new TALE construct and reporter constructs and making transgenic flies and fly crosses. Testing and optimising the quantitative in situ hybridization in these flies

TALE::Bcd constructs will be based on previously generated vectors (Crocker & Stern, 2013). The TALE::Bcd binding site can be added to the rhoNEE sequence and can be increased stepwise to facilitate more TALE::Bcd binding, which augments TF-activity (Crocker et al., 2016; Crocker & Stern, 2017). This will provide a quantitative measure of transcriptional output. rho NEE with TALE-binding sites will be synthesized and packaged in plasmids manufactured by Integrated DNA Technologies (IDT, IA, USA). Gibson assembly will be used for the creation of all the new constructs (Gibson et al., 2009).