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.
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.
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DOI：
10.3390/ijms18122610
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发表时间：
2017-12-04
影响因子：
5.6
通讯作者：
Tanimoto A
中科院分区：
文献类型：
--
作者：
Sato M;Miyoshi K;Nakamura S;Ohtsuka M;Sakurai T;Watanabe S;Kawaguchi H;Tanimoto A
基因组编辑技术的最新进展，如CRISPR/Cas9系统，已经能够通过一步转染分离出具有敲除多个等位基因的细胞。体细胞核移植（SCNT）已被广泛应用于转基因动物生产的有效手段之一。为了使用转基因细胞作为SCNT供体，通常需要在多个目标位点上有效分离具有突变的转染物。分离这种转基因细胞的方法在很大程度上依赖于使用可选择基因的基于药物选择的方法；然而，分离具有多个靶位点突变的细胞通常是困难的。在这项研究中，我们采用了一种新的方法，通过一步引入CRISPR/ cas9相关成分，有效地分离出至少有两个靶位点突变的猪细胞。靶向GGTA1基因的单导RNA （sg）参与细胞表面α-Gal表位（称为异种抗原）的合成，始终是一个先决条件。当转染的细胞与毒素标记的BS-I-B4分离素在37°C下反应2小时以消除表达α-Gal表位的细胞时，存活的克隆缺乏α-Gal表位表达，并且高度期望在另一个靶位点表现出诱导突变。对这些α-Gal表位阴性存活细胞的分析表明，目标位点上100%发生了基因组编辑。使用这些细胞作为供体的SCNT产生了基因型与供体细胞相似的克隆囊胚。因此，这个新系统将有助于scnt介导的转基因克隆仔猪的获取，其中多个目标位点可能发生突变。
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 α-Gal epitope (known as xenogenic antigen), is always a prerequisite. When the transfected cells were reacted with toxin-labeled BS-I-B4 isolectin for 2 h at 37 °C to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. Analysis of these α-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.
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影响因子：
4.7
作者：
Carnero A;Paramio JM
通讯作者：
Paramio JM
影响因子：
3.7
作者：
Li K;Wang G;Andersen T;Zhou P;Pu WT
通讯作者：
Pu WT
影响因子：
4
作者：
Heo, Young Tae;Quan, Xiaoyuan;Kim, Jongpil
通讯作者：
Kim, Jongpil
DOI：
10.1073/pnas.0307819101
发表时间：
2004-05-11
影响因子：
11.1
作者：
Kolber-Simonds, D;Lai, LX;Hawley, RJ
通讯作者：
Hawley, RJ
影响因子：
2.5
作者：
Fujimura, Tatsuya;Takahagi, Yoichi;Murakami, Hiroshi
通讯作者：
Murakami, Hiroshi