title: Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain.
abstract: Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Yuji
authors: Y
authors: Tsunekawa
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Raymond Kunikane
authors: RK
authors: Terhune
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Ikumi
authors: I
authors: Fujita
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Atsunori
authors: A
authors: Shitamukai
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors: Taeko
authors: T
authors: Suetsugu
authors: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan fumio@cdb.riken.jp.
authors: Fumio
authors: 0000-0001-7902-4520
authors: F
authors: Matsuzaki
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology; D018274:Electroporation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: CAS9; CRISPR; Ferret; Gene knock-in; In utero electroporation; Lineage tracing; Mouse