Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System
| Autor: | Takayuki Sakurai, Masato Ohtsuka, Akihide Tanimoto, Hiroaki Kawaguchi, Satoshi Watanabe, Shingo Nakamura, Masahiro Sato, Kazuchika Miyoshi |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
α-Gal epitope p53 Nuclear Transfer Techniques alpha-1 3-galactosyltransferase PTEN Swine α-1 3-galactosyltransferase Biology Catalysis Epitope Article alpha-Gal epitope lcsh:Chemistry Inorganic Chemistry Animals Genetically Modified 03 medical and health sciences targeted toxin Genome editing Antigen Lectins CRISPR Animals genome editing Physical and Theoretical Chemistry isolectin BS-I-B4 lcsh:QH301-705.5 Molecular Biology Gene CRISPR/Cas9 Spectroscopy Alleles Genetics Gene Editing Cas9 isolectin BS-I-B-4 Organic Chemistry General Medicine Transfection Galactosyltransferases Computer Science Applications 030104 developmental biology LDLR lcsh:Biology (General) lcsh:QD1-999 Mutation Somatic cell nuclear transfer CRISPR-Cas Systems |
| Zdroj: | International Journal of Molecular Sciences; Volume 18; Issue 12; Pages: 2610 International Journal of Molecular Sciences International Journal of Molecular Sciences, Vol 18, Iss 12, p 2610 (2017) |
| ISSN: | 1422-0067 |
| DOI: | 10.3390/ijms18122610 |
| Popis: | The recent advancement in genome editing such a CRISPR/Cas9 system has enabled isolation of cells with knocked multiple alleles through a one-step transfection. Somatic cell nuclear transfer (SCNT) has been frequently employed as one of the efficient tools for the production of genetically modified (GM) animals. To use GM cells as SCNT donor, efficient isolation of transfectants with mutations at multiple target loci is often required. The methods for the isolation of such GM cells largely rely on the use of drug selection-based approach using selectable genes; however, it is often difficult to isolate cells with mutations at multiple target loci. In this study, we used a novel approach for the efficient isolation of porcine cells with at least two target loci mutations by one-step introduction of CRISPR/Cas9-related components. A single guide (sg) RNA targeted to GGTA1 gene, involved in the synthesis of cell-surface alpha-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B-4 isolectin for 2 h at 37 degrees C to eliminate alpha-Gal epitope-expressing cells, the surviving clones lacked alpha-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these alpha-Gal epitope-negative surviving cells demonstrated a 100% occurrence of genome editing at target loci. SCNT using these cells as donors resulted in the production of cloned blastocysts with the genotype similar to that of the donor cells used. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets, in which multiple target loci may be mutated. Article INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES.18(12):2610(2017) |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|