文章摘要
徐 帜,孙文博,张妍妍,许 勇,唐金海.利用CRISPR/Cas9技术建立RelB敲除小鼠模型[J].南京医科大学学报,2019,(3):313~319
利用CRISPR/Cas9技术建立RelB敲除小鼠模型
Establishment of RelB knockout mouse models by the CRISPR/Cas9 system
投稿时间:2018-04-18  
DOI:10.7655/NYDXBNS20190301
中文关键词: RelB  CRISPR/cas9  基因敲除  C57BL/6小鼠
英文关键词: RelB  CRISPR/cas9  gene knockout  C57BL/6 mice
基金项目:国家自然科学基金(81872365);国家重点研发计划(2016YFC0905900)
作者单位
徐 帜 南京医科大学第一临床医学院江苏 南京 210029 
孙文博 南京医科大学附属肿瘤医院江苏 南京 210009 
张妍妍 南京医科大学附属肿瘤医院江苏 南京 210009 
许 勇 南京医科大学附属肿瘤医院江苏 南京 210009 
唐金海 南京医科大学第一临床医学院江苏 南京 210029 
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中文摘要:
      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。
英文摘要:
      Objective:To evaluate the relationship between the nuclear factor?κB(NF?κB)family member RelB and tumor metastasis,we established a RelB knockout mouse model by CRISPR/Cas9 system. Methods:The RelB specific single?guide RNAs targeted exon4 were designed for CRISPR/Cas9 system. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase and microinjected into the mouse zygote. C57BL/6 mouse fertilized eggs injected with sgRNA and Cas9 mRNA in vitro were transplanted into mouse uterus. F1 generation was obtained by mating F0 generation with wild type. PCR and gene sequencing were performed to identify the RelB phenotype of F0 and F1 generation mice. The expression of RelB in F0 and F1 generation mice was evaluated in gene level and protein level. Results:We obtained four RelB-/+ F0 mice and stable inherited RelB-/- offspring. The results of genome sequencing showed that there was a nonsense mutation in the mRNA of RelB in the knockout group,which caused the mRNA translation to terminate. Compared with the control group,the expression of Relb protein was not detected in the knockout group. Meanwhile,the results of Western blot showed that there was no significant difference between knockout group and control group in the expression of NF?κB family members(RelA,p50 and p52). Conclusion:The RelB knockout C57BL/6 mouse model was successfully established,which provided an important tool for the study of RelB in tumor metastasis and drug resistance.
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