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Molecular, Biochemical, and Genetic Techniques

開催日 2014/9/11
時間 16:00 - 17:00
会場 Poster / Exhibition(Event Hall B)

Simple and efficient generation of knock-in transgenic zebrafish using CRISPR/Cas9 system

  • P1-388
  • 木村 有希子 / Yukiko Kimura:1 東島 眞一 / Shin-ichi Higashijima:1,2 
  • 1:自然科学研究・岡崎統合バイオ・神経分化 / Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan 2:自然科学研究・生理研 / National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan 

The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. In zebrafish, such transgenic animals have generally been generated using BAC (bacterial artificial chromosome) mediated transgenic methods. However, making BAC DNA constructs are time-consuming. Furthermore, it is not always possible to generate transgenic fish that fully recapitulate endogenous gene expression even with BAC constructs. If efficient knock-in methods are established in zebrafish, it would be of great benefit. Quite recently, Auer et al have shown that knock-in zebrafish could be generated using CRISPR/Cas9system (Genome Research, 2014). In this method, concurrent digestion of the genemic DNA and the donor plasmid with CRISPR/Cas9 resulted in incorporation of the donor plasmid into the genome via a non-homologous recombination pathway. Here, we have modified this method, and succeeded in generating knock-in transgenic zebrafish having reporter gene or Gal4 gene expression that mimics endognenous gene expression. The method is simple, and the efficiency of obtaining transgenic founder was very high (over 20 %). We propose that CRISPR/Cas9-mediated knock-in will become a standard method to generate transgenic lines expressing reporter gene or Gal4 gene expression in a tissue or cell-type specific manner.

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