Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases
Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases
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DOI：
10.1073/pnas.1106422108
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发表时间：
2011-07-19
影响因子：
11.1
通讯作者：
Niemann, Heiner
中科院分区：
文献类型：
--
作者：
Hauschild, Janet;Petersen, Bjoern;Niemann, Heiner
锌指核酸酶(ZFN)是高效产生基因敲除(KO)的有力工具。虽然ZFN介导的基因破坏已在小鼠、大鼠和果蝇等实验动物中得到证实，但ZFN尚未在任何大型家养物种中用于破坏内源基因。在这里，我们使用ZFNS来诱导猪α1，3-半乳糖基转移酶(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处理原代猪成纤维细胞，导致与ZFN处理细胞类似的双等位基因敲除。这些细胞的半乳糖(Gal)表位反选择群体用于体细胞核移植(SCNT)。在得到的6个胎儿中，所有的都完全缺乏Gal表位，并且与最初的供体细胞群体没有表型上的区别，这表明ZFN介导的基因改造没有干扰克隆过程。既没有检测到脱靶切割事件，也没有检测到ZFN编码质粒的整合。补体裂解试验证实了GGTA1-KO的表型，证明了GGTA1-KO成纤维细胞相对于野生型细胞的保护作用。来自GGTA1-KO胎儿的细胞和混合的、转基因的细胞通过SCNT产生活的后代。本研究报告了携带ZFN诱导的内源基因双等位基因敲除的克隆猪的生产。这些发现为创造基因KO猪开辟了一条独特的途径，这可能会使农业和生物医学都受益。
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 alpha 1,3-galactosyl-transferase (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 similar to 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 phenotypically 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.