Elucidation of the mechanism of Cre-loxP recombination by direct introduction of Cre recombinase and application to artificial nuclease (TALENs) in Aspergillus oryzae

2015 Fiscal Year Final Research Report

• Project/Area Number: 25871247
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Applied biochemistry; Applied molecular and cellular biology
• Research Institution: National Research Institute of Brewing
• Principal Investigator: Mizutani Osamu 独立行政法人酒類総合研究所, 研究部門, 主任研究員 (10443996)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: 遺伝子工学 / 麹菌 / マーカーリサイクリング / Cre/loxP システム / 人工ヌクレアーゼ (TALEN)

Outline of Final Research Achievements

We developed a simple method to use the Cre-loxP recombination system for marker gene rescue in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme. To apply the simple method to artificial nucleases (TALEN) for genome editing, we examined kinds of the carrier and conditions of Cre without the carrier for direct introduction. In the carrier, RNA and heparin as well as DNA were available for the carrier. We showed that to raise the electric charge and the concentration of Cre was available for the direct introduction without using the carrier. In addition, because the genome editing of A. oryzae via the error of nonhomologous end-joining repair by a transient expression of TALENs was confirmed, the recombinant TALENs were purified. Hence, we will perform the direct introduction for the genome editing of A. oryzae using the purified TALENs.

Free Research Field

農学