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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)

Ultra-rapid generation of Pax6 mutant mice via CRISPR/Cas9-mediated genome engineering

  • P1-386
  • 井上-上野 由紀子 / Yukiko U. Inoue:1 井上 高良 / Takayoshi Inoue:1 
  • 1:国立精神・神経医療研究センター・神経研究所・疾病研究第六部 / Dept Biochem and Cellular Biol, National Institute of Neuroscience, Tokyo, Japan 

The recent fast-growing development of genome editing technologies have opened the window for the next generation of targeted mutagenesis. The RNA-guided Cas9 nuclease can introduce a double strand break (DSB) into the targeted genome region which leads to error-prone non-homologous end joining. In mouse one-cell embryos, these steps result in mutant mice carrying insertions/deletions at the cut site.
Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, cortical arealization and so forth. Spontaneous mutations in the mouse Pax6 cause an eyeless phenotype in the homozygous condition.
Here we applied the pX330 plasmid (Cong L et al., Science 2013) to express Cas9 and single-guide RNA (sgRNA). We designed sgRNAs to target Pax6 exon5 coding a part of the paired box domain and utilized the single strand annealing assay that reconstitutes EGFP expression to validate which sgRNA sequence works efficiently (Mashiko D et al., Sci Rep 2013). We directly injected the pX330-sgRNA plasmid DNA into pronuclei of fertilized eggs (Mashiko D et al., Sci Rep 2013) and genotyped the embryos by PCR and sequence analysis. As a result, we found insertions/deletions around the DSB site and more than 75% of the embryos showed eyeless or small eye phenotypes with shorter noses due to the deficient migration of the neural crest cells. The brains of the mutant embryos also lost their olfactory bulbs. These phenotypes were remarkably similar to the existing Pax6 small eye mutant ones.
We could achieve this Pax6 gene knockout within a month via a single pronuclear injection, which is much shorter period and much lower expense than the conventional ES cell-based gene targeting method, and the mutation rate was extremely high. CRISPR/Cas9 mediated ultra-rapid production of gene-targeted mice should hence serve as a powerful tool for the investigation in neurodevelopmental and neuronal disease research area.

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