CRISPR系统特异性敲除目的基因在肝病小鼠模型中的应用  

Target gene knockout byCRISPR system in a mouse model of liver disease

作　　者：马月[1] 莫丽 方杰[1] 张欢欢[1] 余陈欢[1] 

机构地区：[1]浙江省医学科学院动物中心,浙江省实验动物与安全性研究重点实验室,杭州310013 [2]浙江省中医药大学药学院,杭州310053

出　　处：《国际流行病学传染病学杂志》2017年第3期151-155,共5页International Journal of Epidemiology and Infectious Disease

基　　金：浙江省医药卫生科技计划（2015KYA060）;浙江省科技计划（2015C37095）

摘　　要：目的利用腺相关病毒（AAV）转导和CRISPR技术建立肝脏相关疾病小鼠模型。方法AAV-绿色荧光蛋白（GFP）表达系统（AAV-GFP）和CRISPR靶向基因敲除质粒系统（px330-sgGFP—Cas9），三组FVB／NJ小鼠采用尾静脉高压水动力注射技术分别注射生理盐水（对照组）、AAV-GFP（AAV-GFP组）、AAV—GFP＋px330-sgGFP-Cas9（sgGFP组），采用小动物活体成像仪检测小鼠肝脏GFP表达情况。结果生理盐水对照组小鼠肝脏不表达GFP；AAV—GFP组小鼠肝脏72h后开始表达GFP，2周后GFP表达稳定；sgGFP组在第9、12天均表达GFP蛋白，但2周后GFP蛋白明显减弱。结论本研究所构建的AAV—GFP表达系统能够在小鼠肝脏中稳定表达GFP。所构建的CRISPR质粒系统可以特异性敲除小鼠肝脏所携带的目的基因，建立合适的肝脏疾病研究模型。Objective To construct a mouse model with liver-related disease by adeno-associated virus （AAV）- mediated gene transfer technology and CRISPR system. Methods AAV-green fluorescent protein （GFP） system （AAV-GFP） and CRISPR targeted gene knockout plasmid system （px330-sgGFP-Cas9） were constructed. Three groups of FVB/NJ mice were injected physiological saline （control group）, AAV-GFP （AAV-GFP group） and AAV- GFP＋px330-sgGFP-Cas9 （sgGFP group）, respectively, by mouse tail vein hydrodynamic injection. Specific expression of GFP in the livers of mouse models were observed by Clairvivo OPT plus. Results The control group did not express GFP. AAV-GFP group began to express GFP after 72 h, and achieved a stable expression after 2 weeks. The sgGFP group expressed GFP on d9 and d12, and showed a significantly weakened expression after 2 weeks. Conclusions The AAV-GFP system can stably express GFP in the liver of mice. CRISPR plasmid system can be used to knock down the expression of specific genes in mouse liver and establish a useful model of liver diseases.

关 键 词：质粒 CRISPR技术 腺相关病毒系统 水动力注射技术 

分 类 号：R-332[医药卫生] R575