Successful production of genome-edited rats by the rGONAD method.
Successful production of genome-edited rats by the rGONAD method.
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DOI：
10.1186/s12896-018-0430-5
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发表时间：
2018-04-02
影响因子：
3.5
通讯作者：
Matsuyama M
中科院分区：
文献类型：
--
作者：
Kobayashi T;Namba M;Koyano T;Fukushima M;Sato M;Ohtsuka M;Matsuyama M
CRISPR/Cas9系统开发的最新进展已被证明是各种生物中有效的基因编辑技术。我们最近在小鼠中开发了一种称为通过输卵管核酸递送（GONAD）进行基因组编辑的新方法;一种不需要离体处理胚胎的新型体内基因组编辑系统，这项技术是新开发的，并更名为“改进的GONAD”（i-GONAD）。然而，这项技术仅限于老鼠。因此，在这项研究中，我们挑战将这项技术应用于大鼠。在这里，我们确定了最合适的条件，在体内基因传递对大鼠植入前胚胎使用四甲基罗丹明标记的葡聚糖，称为大鼠改善的GONAD（rGONAD）。然后，为了研究这种方法是否可以通过递送CRISPR/Cas9组分来产生基因组编辑的大鼠，酪氨酸酶（Tyr）基因被用作靶标。一些幼仔表现出白化色的皮毛，表明野生型Tyr基因等位基因的破坏。此外，我们确认rGONAD方法可以用于通过基于ssODN的敲入在大鼠基因组中引入遗传改变。我们首先建立了rGONAD方法来产生基因组编辑的大鼠。我们证明了rGONAD方法生产敲除和敲入大鼠的高效率，这将有助于大鼠基因组工程实验的生产。rGONAD方法也可以容易地应用于哺乳动物，例如豚鼠、仓鼠、牛、猪和其它哺乳动物。本文的在线版本（10.1186/s12896-018-0430-5）包含补充材料，可供授权用户使用。
Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD” (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals. The online version of this article (10.1186/s12896-018-0430-5) contains supplementary material, which is available to authorized users.
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影响因子：
7.3
作者：
SADO, Y;KAGAWA, M;OKIGAKI, T
通讯作者：
OKIGAKI, T
影响因子：
4.6
作者：
Takahashi G;Gurumurthy CB;Wada K;Miura H;Sato M;Ohtsuka M
通讯作者：
Ohtsuka M
影响因子：
3.7
作者：
Kaneko T;Mashimo T
通讯作者：
Mashimo T
影响因子：
4.6
作者：
Kaneko T;Sakuma T;Yamamoto T;Mashimo T
通讯作者：
Mashimo T
影响因子：
3.6
作者：
NUTI, KM;SRIDHARAN, BN;MEYER, RK
通讯作者：
MEYER, RK