The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.
The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.
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DOI：
10.1371/journal.pone.0142946
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Gurdon JB
中科院分区：
文献类型：
--
作者：
Miyamoto K;Suzuki KT;Suzuki M;Sakane Y;Sakuma T;Herberg S;Simeone A;Simpson D;Jullien J;Yamamoto T;Gurdon JB
使用可编程核酸酶进行基因组编辑的最新进展已经彻底改变了各种生物体中的基因靶向。成功的基因敲除已被证明在非洲爪蟾，一个广泛使用的模式生物，虽然一个系统，使更少的马赛克敲除的创始人胚胎（F0）需要进行探索，以判断表型的F0代。在这里，我们注射修改后的高活性转录激活因子样效应核酸酶（TALEN）的mRNA卵母细胞在生殖囊泡（GV）阶段，然后在体外成熟和胞浆内单精子注射，以实现在F0胚胎的完全敲除。与传统的受精胚胎注射方法不同，将TALEN mRNA注射到GV卵母细胞中可以在受精前表达核酸酶，使它们能够在早期阶段发挥作用。使用此程序，大多数发育的胚胎显示完全敲除表型的色素沉着基因酪氨酸酶和/或胚胎致死基因pax 6在创始人一代。此外，我们的方法允许一个大的1 kb的删除。因此，我们描述了非洲爪蟾F0胚胎中几乎完全的基因敲除表型。所提出的方法将有助于加快生产敲除青蛙，因为我们可以绕过约1年的额外一代非洲爪蟾。同时，我们的方法提供了一个独特的机会，可以快速测试破坏那些不允许生长到能够繁殖的成年人的基因的发育影响。此外，这里所示的协议是相当少的侵入性比以前使用的主机转移，因为我们的协议不需要手术。提出的实验方案是潜在的适用于其他生物，如哺乳动物和鱼类，以解决常见的问题，镶嵌在创始人。
Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.