A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes
A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes
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一种利用体外受精卵产生 KO 大鼠的简单有效方法
DOI：
10.1007/978-1-0716-3016-7_18
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发表时间：
2023
影响因子：
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通讯作者：
Masahide Asano
中科院分区：
文献类型：
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作者：
Kohtaro Morita;Arata Honda;Masahide Asano
ZFN、TALEN和CRISPR/Cas9系统的开发简化了除小鼠外产生敲除（KO）和敲入（KI）大鼠的过程。然而，在大鼠中，尚未建立使用体外受精卵母细胞的有效基因组编辑技术。最近，我们报道了五个标准品系大鼠通过超数排卵和体外受精（IVF）获得稳定后代。此外，基因组编辑的大鼠可以很容易地通过电穿孔产生。首先，给幼年雌性大鼠施用LHRH（促黄体生成素释放激素）以同步发情周期，然后在hCG（人绒毛膜促性腺激素）之前用PMSG（孕马血清促性腺激素）给AIS（自动识别系统）进行超数排卵。然后将第二天早上从性成熟的雄性大鼠收集的精子进行预培养。从麻醉下的雌性大鼠收集卵丘细胞-卵母细胞复合体（COC），并将COC诱导到含有浓度调节的精子的培养基中。此后，选择具有两个原核的卵母细胞作为受精卵母细胞。接下来，将受精的卵母细胞转移到含有由gRNA和Cas9蛋白形成的CRISPR核糖核蛋白（RNP）复合物的玻璃室中。电穿孔后，立即将受精的卵母细胞转移到培养基中。第二天，将胚胎移植到假孕雌性大鼠的输卵管中。使用上述方法，胚胎移植当天22天后即可获得后代。在本文中，我们概述了一种方法，允许简单而有效地生成转基因大鼠，而不需要技术上困难的显微操作技术。
The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation. Sperm collected from a sexually mature male rat the following morning is then pre-cultured. Cumulus cell-oocyte complexes (COCs) are collected from female rats under anesthesia, and COCs are induced into a medium containing concentration-adjusted sperm. Thereafter, oocytes with two pronucleus are selected as fertilized oocytes. Next, fertilized oocytes are transferred into a glass chamber containing CRISPR ribonucleoprotein (RNP) complexes formed from gRNA and Cas9 protein. After electroporation, fertilized oocytes are then immediately transferred to culture medium. The next day, embryos are transferred into the oviduct of pseudopregnant female rats. Using the above method, offspring can be obtained 22 days after the day of embryo transfer. In this paper, we outline a method allowing simple and efficient generation of genetically modified rats without the need for technically difficult micromanipulation techniques.