Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells.

Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians & Surgeons, New York, NY 10032.
Science (Impact Factor: 31.48). 12/1989; 246(4931):799-803. DOI: 10.1126/science.2554496
Source: PubMed

ABSTRACT A substitution mutation has been introduced into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene, and these cells have been used to generate chimeric mice. It is shown that the c-abl mutation was transmitted to progeny by several male chimeras. This work demonstrates the feasibility of germ-line transmission of a mutation introduced into a nonselectable autosomal gene by homologous recombination.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Conventional embryonic stem cell (ESC)-based gene targeting, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
    Nature Protocols 10/2014; 9(10):2493-2512. DOI:10.1038/nprot.2014.171 · 7.78 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.
    In Vitro Cellular & Developmental Biology - Animal 12/2014; 51(1). DOI:10.1007/s11626-014-9805-7 · 1.00 Impact Factor
  • Source