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首页> 外文期刊>Biology Open >Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice
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Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

机译：超超排卵，用于CRISPR-Cas9介导的基因敲除，单氨基酸取代的和成群的小鼠生产

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Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via?in vitro?fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.
机译：使用基因组编辑技术生产转基因小鼠的最新进展表明，需要改善限制因素，包括显微注射的合子收集和冷冻保存。最近，我们开发了一种新的超排卵技术，使用抑制素抗血清和马绒毛膜促性腺激素来促进卵泡生长。与传统的超排卵方法相比，该方法能够通过体外受精增加受精卵的产量。在这里，我们验证了超超排卵技术可用于通过将质粒载体或核糖核蛋白显微注射到受精卵中，有效地产生簇状规则间隔的短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）介导的敲除小鼠。我们还调查了是否可以生成单氨基酸取代的小鼠和条件性基因敲除小鼠。通过共显微注射Cas9蛋白，指导RNA和单链寡聚脱氧核苷酸（ssODN），可以比通过ssODN质粒显微注射更有效地产生带有碱基取代的Founder小鼠。有条件的等位基因通过一步插入一个ssODN成功引入，该ssODN被设计为携带通过两个loxP序列和同源臂使用双切CRISPR-Cas9策略侧翼的外显子。从动物福利和工作效率的角度出发，我们的研究提出了一种基于CRISPR-Cas9的转基因小鼠的有用方法。

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