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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases
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Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

机译：使用锌指核酸酶在猪中高效产生双等位基因敲除

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摘要

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyl-transf erase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1. resulting in biallelic knockout of ~1 % of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were pheno-typically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.
机译：锌指核酸酶（ZFN）是高效产生基因敲除（KO）的有力工具。 ZFN介导的基因破坏已在实验动物如小鼠，大鼠和果蝇中得到证实，而ZFN尚未用于破坏任何大型家养物种中的内源基因。在这里，我们使用ZFNs诱导猪α1,3-半乳糖基-转移擦除（GGTA1）基因的双等位基因敲除。用针对GGTA1催化核心编码区设计的ZFN处理原始猪成纤维细胞。导致双等位基因敲除〜1％的ZFN处理细胞。这些细胞的半乳糖（Gal）表位反选群体用于体细胞核移植（SCNT）。在产生的六个胎儿中，所有胎儿都完全缺乏Gal表位，并且与起始供体细胞群体在表型上没有区别，这说明ZFN介导的遗传修饰不会干扰克隆过程。既未检测到脱靶裂解事件，也未检测到ZFN编码质粒的整合。通过补体裂解测定法证实了GGTA1-KO表型，其证明了GGTA1-KO成纤维细胞相对于野生型细胞的保护。来自GGTA1-KO胎儿的细胞和合并的转染细胞用于通过SCNT产生活后代。这项研究报告了携带双等位基因ZFN诱导的内源基因敲除的克隆猪的生产。这些发现为基因KO猪的创造开辟了一条独特的途径，这可以使农业和生物医学都受益。

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    Janet Hauschild; Bjoern Petersen; Yolanda Santiago; Anna-Lisa Queisser; Joseph W. Carnwath; Andrea Lucas-Hahn; Lei Zhang; Xiangdong Meng; Philip D. Gregory; Reinhard Schwinzer; Gregory J. Cost; Heiner Niemann;

  • 作者单位

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany;

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany;

    Sangamo BioSciences, Richmond,CA 94804;

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany;

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany Rebirth, Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany;

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany;

    Sangamo BioSciences, Richmond,CA 94804;

    Sangamo BioSciences, Richmond,CA 94804;

    Sangamo BioSciences, Richmond,CA 94804;

    Transplantation Laboratory, Hannover Medical School, 30625 Hannover, Germany;

    Sangamo BioSciences, Richmond,CA 94804;

    Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, 31535 Neustadt am Rubenberge, Germany Rebirth, Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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  • 正文语种 eng
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  • 关键词

    fetal fibroblasts; xenotransplantation; off target sites; complement mediated lysis assay;

    机译：胎儿成纤维细胞异种移植偏离目标地点;补体介导的裂解测定;

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