Development of screening system for bone forming reagents by using TALEN genome engineering technique

2015 Fiscal Year Final Research Report

• Project/Area Number: 26670807
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Functional basic dentistry
• Research Institution: Osaka University
• Principal Investigator: Nishimura Riko 大阪大学, 歯学研究科(研究院), 教授 (60294112)
• Co-Investigator(Renkei-kenkyūsha): HATA KENJI 大阪大学, 大学院歯学研究科, 准教授 (80444496) ; MURAKAMI TOMOHIKO 大阪大学, 大学院歯学研究科, 講師 (50510723)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 骨形成 / ゲノム編集

Outline of Final Research Achievements

In order to develop effective screening system for bone forming reagents, we attempted to generate cell lines knock-ined both Venus and luciferase genes by using the Golden Gate TALEN genome engineering technique. Although the Golden Gate TALEN genome engineering technique is much more effective and useful than conventional TALEN system, selection of positive clone cells was still complicate. We, therefore, attempted to optimize a new genome engineering technique, PITCh method, which would be very powerful for knock-in of interested genes into to mammalian cells. First, we used Cas9-based PITCh technique, allowing us to generate guide RNA construct easily. Second, we designed and generated a PITCh donor vector, in which we introduced CLuc luciferase, Puromycin resistant genes with EGFP expression cassette. This optimization seems very effective and helpful for selection of the positive clone cells.

Free Research Field

分子生物学