i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats
i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats
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DOI：
10.1038/s41598-018-30137-x
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发表时间：
2018-08
影响因子：
4.6
通讯作者：
Shuji Takabayashi;T. Aoshima;K. Kabashima;Kazushi Aoto;M. Ohtsuka;Masahiro Sato
中科院分区：
文献类型：
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作者：
Shuji Takabayashi;T. Aoshima;K. Kabashima;Kazushi Aoto;M. Ohtsuka;Masahiro Sato
受精卵显微注射或分离受精卵的体外电穿孔是目前广泛使用的产生基因组编辑小鼠的方法。然而，这些技术需要费力和耗时的受精卵活体处理，包括合子分离、基因转移到受精卵和胚胎移植到受精者体内。我们最近开发了一种替代方法，称为通过输卵管核酸传递改进的基因组编辑(I-GONAD)，它不需要上述的受精卵活体处理，而是在1-细胞胚胎晚期在解剖显微镜下和随后的电穿孔下，将基因组编辑组件、Cas9蛋白和合成的gRNA注入到怀孕雌性的输卵管中。用这种方法，我们成功地以相对较高的效率产生了基因组编辑的小鼠(例如，以~97%的效率产生了敲除等位基因)。在这里，我们将这项改进的技术推广到老鼠身上，发现I-性腺可以创造出各种品系的基因组编辑大鼠，包括SpragueDawley和Lewis，以及F1杂交后代(SpragueDawley和Brown挪威)，其插入突变和敲入突变的效率分别为62%和9%。因此，i-性腺对于在缺乏昂贵的显微操纵器系统和高技能的胚胎操作人员的小型实验室中生产基因组编辑的大鼠将特别有用。
Zygote-microinjection orin vitroelectroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consumingex vivohandling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentionedex vivohanding of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found thati-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus,i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.