Sato Masahiro, Nakamura Ayaka, Sekiguchi Marie, Matsuwaki Takashi, Miura Hiromi, Gurumurthy Channabasavaiah B, Kakuta Shigeru, Ohtsuka Masato
Department of Genome Medicine, National Center for Child Health and Development, Tokyo, Japan.
Support Center for Medical Research and Education, Tokai University, Kanagawa, Japan.
Methods Mol Biol. 2023;2631:325-340. doi: 10.1007/978-1-0716-2990-1_14.
The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.
成簇规律间隔短回文重复序列（CRISPR）技术使生产基因编辑（GE）动物比以往更容易、更迅速。在大多数情况下，基因编辑小鼠是通过将CRISPR试剂显微注射（MI）到受精卵（合子）中或通过体外电穿孔（EP）来产生的。这两种方法都需要对分离的胚胎进行离体操作，并随后将其转移到另一组小鼠（称为受体或假孕小鼠）体内。此类实验由技术娴熟的技术人员进行（尤其是显微注射）。我们最近开发了一种新的基因编辑方法，称为“GONAD（通过输卵管核酸递送进行基因编辑）”，该方法可以完全消除胚胎的离体操作。我们还对GONAD方法进行了改进，称为“改进型GONAD（i-GONAD）”。i-GONAD方法包括在解剖显微镜下使用口控玻璃微量移液器将CRISPR试剂注射到麻醉的怀孕雌性动物的输卵管中，然后对整个输卵管进行电穿孔，使CRISPR试剂原位进入输卵管内的合子中。在i-GONAD操作后，让从麻醉中恢复的小鼠继续怀孕至足月以产下幼崽。与依赖合子离体操作的方法不同，i-GONAD方法不需要假孕雌性动物来进行胚胎移植。因此，与传统方法相比，i-GONAD方法可以减少所用动物的数量。在本章中，我们描述了一些关于i-GONAD方法的更新技术提示。此外，尽管GONAD和i-GONAD的详细方案已在其他地方发表（Gurumurthy等人，《人类遗传学实验指南》88:15.8.1 - 15.8.12，2016；《自然实验指南》14:2452 - 2482，2019），但我们在本章中提供了i-GONAD的所有方案步骤，以便读者可以在一个地方找到进行i-GONAD实验所需的大部分信息。
