Gene Targeting in Rabbits: Single-Step Generation of Knockout Rabbits by Microinjection of CRISPR/Cas9 Plasmids
Gene Targeting in Rabbits: Single-Step Generation of Knockout Rabbits by Microinjection of CRISPR/Cas9 Plasmids
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家兔基因打靶：通过显微注射 CRISPR/Cas9 质粒一步生成敲除家兔
DOI：
10.1007/978-1-0716-3016-7_20
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发表时间：
2023
影响因子：
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通讯作者：
Arata Honda
中科院分区：
文献类型：
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作者：
高桑央;廣瀬哲郎;Kurumizaka Hitoshi;Arata Honda
基因组编辑技术的发展使基因破坏在各种动物物种中得以实现，并对许多哺乳动物有益。利用多能干细胞对兔子进行基因破坏很难实现，但由于基因组编辑技术的进步，已经进行了许多基因破坏。本章描述了一种使用CRISPR/Cas9在家兔中进行基因破坏的简单易行的方法，该方法标记待破坏的基因，检查脱靶候选基因的存在与否，并构建一个允许Cas9和sgRNA同时表达的质粒。接下来，研究候选序列的切割活性，并进行评估以确定目标序列是否可以被切割。经超排卵处理的母兔与公兔交配，收集受精卵，进行质粒DNA原核注射。第二天，将两细胞阶段的胚胎移植到假孕兔体内，后代在大约29-30天内出生。然后检查后代的基因组DNA，以检查发生了哪种类型的遗传修饰。随着CRISPR/Cas9的出现，家兔基因破坏的可及性显著提高。本章具体总结了如何在家兔中进行基因破坏。
The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This chapter describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into a pseudopregnant rabbits, and offspring are born within approximately 29–30 days. The genomic DNA of the offspring is then examined to check what type of genetic modifications has occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This chapter summarizes specifically how to carry out gene disruptions in rabbits.