Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease
Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease
复制标题
使用锌指核酸酶敲除猪体细胞中的靶基因
DOI：
10.1111/asj.12259
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发表时间：
2015
影响因子：
2
通讯作者：
Kashiwazaki N
中科院分区：
文献类型：
--
作者：
Hisamatsu S;Sakaue M;Takizawa A;Kato T;Kamoshita M;Ito J;Kashiwazaki N
靶向基因组编辑是一种广泛适用的有效修饰动物中任何感兴趣序列的方法。迄今为止，除小鼠外，很难产生敲除和敲入动物。最近，已经开发了一种使用锌指核酸酶（ZFN）的基因组编辑方法来产生敲除大鼠。由于只需要将ZFN注射到原核（PN）胚胎中，因此它似乎可用于产生基因靶向动物，包括家养物种。然而，还没有人报道通过将ZFN直接注射到PN胚胎中成功生产基因敲除猪。作为初步研究，我们在来自猪的两种细胞系（ST和PT‐K75）中检查了ZFN是否对猪生长激素受体的基因组进行编辑。我们的数据表明，pZFN 1/2载体被有效地转染到ST和PT-K75细胞中。在这两种细胞系中，Cel‐I测定的结果表明，证实了靶基因的修饰。我们将ZFN 1/2基因注射到PN期胚胎的细胞核中，然后将它们转移到受体中。但是，未交付幼仔。总的来说，ZFN甚至可以成为猪的基因组编辑技术，但需要进一步改进以产生基因组修饰的猪。
Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock‐out and knock‐in animals except for mice up to now. Very recently, a method of genome editing using zinc‐finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene‐targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT‐K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT‐K75 cells. In both cell lines, results from Cel‐I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome‐modified pigs.