Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes
Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes
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通过输卵管核酸传递 (i-GONAD) 改进基因组编辑：实验步骤和附加说明
DOI：
10.1007/978-1-0716-2990-1_14
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Ohtsuka Masato
中科院分区：
文献类型：
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作者：
Sato Masahiro;Nakamura Ayaka;Sekiguchi Marie;Matsuwaki Takashi;Miura Hiromi;Gurumurthy Channabasavaiah B.;Kakuta Shigeru;Ohtsuka Masato
成簇的规则间隔短回文重复序列（CRISPR）技术使生产基因组编辑（GE）动物比以前更容易和更快成为可能。在大多数情况下，GE小鼠通过显微注射（MI）或通过CRISPR试剂体外电穿孔（EP）进入受精卵（合子）来产生。这两种方法都需要离体处理分离的胚胎，然后将其转移到另一组小鼠（称为受体或假孕小鼠）中。这些实验由高度熟练的技术人员进行（尤其是MI）。我们最近开发了一种新的基因组编辑方法，称为“GONAD（通过输卵管核酸递送进行基因组编辑）”，它可以完全消除胚胎的体外处理。我们还对GONAD方法进行了改进，称为“改进的GONAD（i-GONAD）”。i-GONAD方法涉及在解剖显微镜下使用接口控制的玻璃微量移液管将CRISPR试剂注射到麻醉的妊娠女性的输卵管中，然后对整个输卵管进行EP，使CRISPR试剂原位进入输卵管内存在的受精卵中。在i-GONAD程序后，允许从麻醉中恢复的小鼠继续妊娠至足月以分娩其幼崽。i-GONAD方法不需要假孕雌性动物进行胚胎移植，不像依赖于受精卵的体外处理的方法。因此，与传统方法相比，i-GONAD方法可以减少使用的动物数量。在本章中，我们将介绍一些关于i-GONAD方法的新技术提示。此外，即使GONAD和-GONAD的详细方案已经在其他地方发表（Gurumurthy等人，Curr Protoc Genet 88：15.8.1-15.8.12，2016 Nat Protoc 14：2452-2482，2019），我们在本章中提供了GONAD的所有方案步骤，以便读者可以在一个地方找到进行GONAD实验所需的大部分信息。
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 viaOviductalNucleicAcidsDelivery),” which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed “improved-GONAD (i-GONAD).” Thei-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 thei-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. Thei-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, thei-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about thei-GONAD method. Additionally, even though the detailed protocols of GONAD andi-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 ofi-GONAD in this chapter so that the reader can find most of the information, needed for performingi-GONAD experiments, in one place.