Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.
Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.
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DOI：
10.1007/978-1-4939-8831-0_11
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Liu C
中科院分区：
文献类型：
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作者：
Liu Y;Du Y;Xie W;Zhang F;Forrest D;Liu C
条件性基因敲除（cKO）小鼠对于生物医学研究非常有价值，因为它们能够以组织或时间特异性的方式详细分析基因功能。传统的cKO小鼠的制备方法耗时耗力，需要制备大的基因靶向构建体，分离筛选多个ES细胞克隆，将阳性ES细胞克隆注入囊胚中制备嵌合体小鼠，并通过生殖细胞系将目的基因传递给嵌合体小鼠。最近开发的CRISPR技术彻底改变了基因工程生物的创造方式。敲除和敲入小鼠现在可以通过直接向受精卵注射Cas9、sgRNA和供体DNA来制备。理论上，使用两个sgRNA和两个寡核苷酸作为供体，通过同时插入两个loxP位点可以产生cKO小鼠，但实际上一步获得cKO小鼠的概率仍然很低，部分是因为寡核苷酸介导的敲入效率比非同源末端连接（NHEJ）低得多，部分是因为共同时切割一个等位基因中的并置位点常常导致两个切割位点之间的整个片段的缺失。因此，许多实验室倾向于顺序插入两个loxP位点，即，首先产生具有一个loxP的小鼠，然后使用从这些小鼠收集的胚胎插入第二个loxP位点。在本章中，我们描述了我们的程序和时间轴使用这种顺序的方法，使Six6 cKO小鼠线作为其可行性的证明。
Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way genetically engineered organisms are created. Knockout and knockin mice can now be made by directly injecting zygotes with Cas9, sgRNA, and donor DNA. In theory, cKO mice can be generated by simultaneously inserting two loxP sites using two sgRNAs and two oligonucleotides as donors, but in practice the probability of obtaining cKO mice in one step is still very low, partly because the efficiency of oligo-mediated knockin is much lower than non-homologous end joining (NHEJ) and partly because co-cutting juxtaposed sites in one allele at the same time often leads to the deletion of the entire fragment between the two cutting sites. Therefore, many laboratories prefer to insert the two loxP sites sequentially, i.e., generating mice with one loxP first and then use embryos collected from these mice to insert the second loxP site. In this chapter, we describe our procedures and timeline using this sequential method to make a Six6 cKO mouse line as a demonstration of its feasibility.