Highly efficient gene knockout by injection of TALEN mRNA into oocytes and host transfer in Xenopus leavis

2015 Fiscal Year Final Research Report

• Project/Area Number: 25430089
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Laboratory animal science
• Research Institution: Hiroshima University
• Principal Investigator: Nakajima Keisuke 広島大学, 理学(系)研究科(研究院), 助教 (60260311)
• Co-Investigator(Kenkyū-buntansha): YAOITA YOSHIO 広島大学, 理学研究科, 教授 (00166472)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: Host-transfer / TALEN / Genome editing / Targeted gene knockout / Xenopus

Outline of Final Research Achievements

Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs) and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3’UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3’UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf).

Free Research Field

発生生物学