利用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠  

Construction of miRNA-29b1 knockout mice based on CRISPR/Cas9 technology

作　　者：赵勇[1] 师长宏[1] 赵亚[1] 辛智倩 刘佩娟[1] 张彩勤[1] 白冰[1] 白杰英[2] 王华[3] 张海[1] 

机构地区：[1]第四军医大学实验动物中心,西安710032 [2]军事医学科学院实验动物中心,北京100071 [3]安徽医科大学第一附属医院肿瘤科,合肥230022

出　　处：《中国比较医学杂志》2016年第12期1-4,共4页Chinese Journal of Comparative Medicine

基　　金：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。Objective To construct miRNA-29bl gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29bl 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-29bl in the heart, liver, spleen, lung and kidney of mutated mice. Result A 20 hp sgRNA targeted on miRNA-29bl was synthesized and transcribed to RNA with Casg. After mieroinjection, miRNA-29bl gene-mutated mice were obtained. The sequencing results showed that there were two types of geuotype 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-29bl in the heart, liver, spleen, lung and kidney was reduced significantly. Conclusions miRNA- 29bl gene knockout mice are constructed successfully by using CRISPR/Cas9 technology.

关 键 词：CRISPR/Cas9 miRNA-29b1 基因敲除小鼠 

分 类 号：R33[医药卫生—人体生理学]