利用CRISPR/Cas9技术建立RelB敲除小鼠模型

Objective: To knockout the RelB gene in C57BL/6 mice using the CRISPR/Cas9 technology and provide an experimental animal model with the RelB gene knockout for the research on the RelB gene of the nuclear factor (NF)-κB transcription factor protein family in aspects such as tumor metastasis. Methods: Using the gene editing technology CRISPR/Cas9 system, design and construct the sgRNA targeting the 4th exon of the target gene RelB, and simultaneously use T7 RNA polymerase for in vitro transcription of Cas9 mRNA. After in vitro injection of sgRNA and Cas9 mRNA into the fertilized eggs of C57BL/6 mice, perform embryo transfer to achieve knockout of the target gene. After the mice are born, extract DNA and perform PCR identification to obtain the F0 generation. Then mate with the wild type for reproduction, sample and extract DNA, and obtain the F1 generation mice after sequencing analysis and identification, and verify the knockout effect at the protein level and the genomic level respectively. Results: Four founder mice with mutations in the RelB gene were obtained, and mice with a stable knockout of the RelB gene were obtained. The results of genomic sequencing showed that a nonsense mutation occurred in the mRNA of RelB in the knockout experimental group, causing the termination of its mRNA translation. Compared with the control group, the expression of the RelB protein could not be detected in the knockout experimental group. Meanwhile, the Western blot results showed that the protein expressions of other members of the NF-κB family, RelA, p50 and p52, were not affected. Conclusion: C57BL/6 mice with specific knockout of RelB were successfully obtained, providing an important tool for the research on RelB in tumor metastasis and drug resistance.

目的:利用CRISP/Cas9技术敲除C57BL/6小鼠中的RelB基因,为核因子(nuclear factor,NF)-κB转录因子蛋白家族的RelB基因与肿瘤转移等方面的研究提供RelB基因敲除实验动物模型。方法:利用基因编辑技术CRISPR/Cas9系统,设计并构建针对目的基因RelB第4外显子sgRNA,同时利用T7 RNA聚合酶体外转录Cas9 mRNA。取C57BL/6小鼠受精卵体外注射sgRNA和Cas9 mRNA后,进行胚胎移植,实现靶基因敲除。待小鼠出生后提取DNA并进行PCR鉴定,获得F0代,后与野生型交配后繁殖,取样提取DNA,测序分析鉴定后获得F1代小鼠,并在蛋白质水平和基因组水平分别验证敲除效果。结果:获得了4个在RelB基因突变的首建鼠,并得到了稳定遗传的RelB基因敲除小鼠。基因组测序结果示,敲除实验组中RelB的mRNA出现无义突变,令其mRNA翻译终止。与对照组相比,敲除实验组检测不到RelB蛋白表达。同时,Western blot结果显示,NF-κB家族中其他成员RelA、p50和p52的蛋白表达不受影响。结论:成功获得特异性敲除RelB的C57BL/6小鼠,为RelB在肿瘤转移、耐药中的研究提供了重要工具。