Animal Resource Laboratory, Center for Development of Advanced Medical Technology, Jichi Medical University School of Medicine, Yakushiji, Shimotsuke-shi, Tochigi, Japan.
Methods Mol Biol. 2023;2637:255-267. doi: 10.1007/978-1-0716-3016-7_20.
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.
基因组编辑技术的发展使得在各种动物物种中实现基因敲除成为可能，并对许多哺乳动物有益。使用多能干细胞进行基因敲除在兔子中很难实现，但由于基因组编辑技术的进步，已经进行了一些基因敲除。本章描述了一种使用 CRISPR/Cas9 在兔子中进行基因敲除的简单易行的方法，其中标记了要敲除的基因，检查了是否存在脱靶候选物，并构建了允许同时表达 Cas9 和 sgRNA 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。经过超数排卵处理的雌性兔子与雄性兔子交配并收集受精卵，然后进行质粒 DNA 的原核注射。第二天，将两细胞期胚胎移植到假孕兔子中，大约 29-30 天后产下后代。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，兔子中基因敲除的可及性显著提高。本章专门总结了如何在兔子中进行基因敲除。
