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CRISPR/Cas9-based simple transgenesis in Xenopus laevis

Shibata, Yuki ; Suzuki, Miyuki ; Hirose, Nao ; Takayama, Ayuko ; Sanbo, Chiaki ; Inoue, Takeshi ; Umesono, Yoshihiko ; Agata, Kiyokazu ; Ueno, Naoto ; Suzuki, Ken-ichi T. ; Mochii, Makoto

Developmental biology, 2022-09, Vol.489, p.76-83 [Periódico revisado por pares]

United States: Elsevier Inc

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  • Título:
    CRISPR/Cas9-based simple transgenesis in Xenopus laevis
  • Autor: Shibata, Yuki ; Suzuki, Miyuki ; Hirose, Nao ; Takayama, Ayuko ; Sanbo, Chiaki ; Inoue, Takeshi ; Umesono, Yoshihiko ; Agata, Kiyokazu ; Ueno, Naoto ; Suzuki, Ken-ichi T. ; Mochii, Makoto
  • Assuntos: CRISPR/Cas9 ; Target integration ; tgfbr2like ; Transgenesis ; Xenopus laevis
  • É parte de: Developmental biology, 2022-09, Vol.489, p.76-83
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
  • Descrição: Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis. Schematic of simple target gene integration using the CRISPR/Cas9 system. The prepared NEXTrans vector including 665 bp of tgfbr2l.L, a selective promoter, and fluorescent gene was co-injected into fertilized Xenopus laevis eggs with Cas9 RNP targeting a common sgRNA designed for tgfbr2l exon4. The plasmid vector integrated into tgfbr2l.L and/or the tgfbr2l.S locus in a forward and/or reverse direction via NHEJ. [Display omitted] •tgfbr2l gene disruption by Cas9 RNP caused no developmental defects.•Cas9 RNP allowed efficient target integration of donor vector at the tgfbr2l loci.•Germline transmission was observed in the F1 offspring.•tgfbr2l loci is a safe harbor site for transgenesis in Xenopus laevis.
  • Editor: United States: Elsevier Inc
  • Idioma: Inglês
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