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利用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠的研究
引用本文:张海,王华,赵勇,师长宏,赵亚,辛智倩,刘佩娟,张彩勤,白冰,白杰英.利用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠的研究[J].中国比较医学杂志,2016,26(12):1-4.
作者姓名:张海  王华  赵勇  师长宏  赵亚  辛智倩  刘佩娟  张彩勤  白冰  白杰英
作者单位:第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;第四军医大学实验动物中心, 西安 710032;军事医学科学院实验动物中心, 北京 100071;安徽医科大学第一附属医院肿瘤科, 合肥 230022;第四军医大学实验动物中心, 西安 710032
基金项目:国家自然科学基金优秀青年基金项目(NO.81522009);国家自然科学基金面上项目(NO.8137257,81272385);军队重点项目(NO.BWS14J058)。
摘    要:目的 应用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠。方法 针对miRNA-29b1基因设计一段sgRNA,sgRNA和Cas9体外转录后显微注射至C57BL/6小鼠受精卵细胞。小鼠出生后取其基因组DNA进行测序以鉴定基因型,同时取小鼠心、肝、脾、肺、肾等脏器研磨后提取总RNA,通过real-time PCR分析miRNA-29b1在这些脏器中的表达。结果 设计了20 bp的miRNA-29b1sgRNA并与Cas9一起进行了体外转录,显微注射小鼠受精卵细胞后获得miRNA-29b1基因突变小鼠。测序结果表明突变小鼠有两种基因型,一种为10 bp的缺失突变;另一种为22 bp的缺失突变,同时伴有3 bp的插入突变。与野生型小鼠相比,基因突变小鼠心、肝、脾、肺、肾等组织中miRNA-29b1表达量下降明显。结论 应用CRISPR/Cas9技术成功构建miRNA-29b1基因敲除小鼠。

关 键 词:CRISPR/Cas9  miRNA-29b1  基因敲除小鼠
收稿时间:2016/5/5 0:00:00
修稿时间:2016/5/13 0:00:00

Construction of miRNA-29b1 knockout mice based on CRISPR/Cas9 technology
ZHAO Yong,SHI Chang-hong,ZHAO Y,XIN Zhi-qian,LIU Pei-juan,ZHANG Cai-qin,BAI Bing,BAI Jie-ying,WANG Hua and ZHANG Hai.Construction of miRNA-29b1 knockout mice based on CRISPR/Cas9 technology[J].Chinese Journal of Comparative Medicine,2016,26(12):1-4.
Authors:ZHAO Yong  SHI Chang-hong  ZHAO Y  XIN Zhi-qian  LIU Pei-juan  ZHANG Cai-qin  BAI Bing  BAI Jie-ying  WANG Hua and ZHANG Hai
Institution:Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China;Laboratory Animal Center, Academy of Military Medical Sciences, Beijing 100071;Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022;Laboratory Animal Center, The Fourth Military Medical University, Xi''an 710032, China
Abstract:Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank. sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C57BL/6 mice. After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile, real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice. Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas9. After microinjection, miRNA-29b1 gene-mutated mice were obtained. The sequencing results showed that there were two types of genotype for the mutated mice, one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion. Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly. Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR/Cas9 technology.
Keywords:CRISPR/Cas9  miRNA-29b1  Gene knockout mice
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