限制性内切酶策略鉴定CRISPR/Cas9介导的Chrm3基因敲除小鼠  

Characterize CRISPR/Cas9 Established Chrm3 Gene Knockout Mice by Enzyme Restriction

作　　者：高旭[1] 马宁[1] 王大勇[1] 张赫[1] 张艳芬[2] 徐亚[1] 贺军[1] 徐书会 霍亚丽[1] 杜智敏[3] 

机构地区：[1]哈尔滨医科大学基础医学院生物化学与分子生物学教研室,哈尔滨150081 [2]哈尔滨医科大学附属第二临床医学院实验诊断教研室,哈尔滨150081 [3]哈尔滨医科大学附属第二医院临床药学药物研究所,哈尔滨150081

出　　处：《中国生物化学与分子生物学报》2016年第4期404-409,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：哈尔滨医科大学于维汉院士杰出青年培养基金(2014);黑龙江省博士后科研启动金(No.LBH-Q15108);高等学校博士学科点专项科研基金项目(No.20122307110002);国家自然科学基金项目(No.81570534)~~

摘　　要：CRISPR/Cas9技术是近年发展起来的快速基因编辑技术。通过该技术已对多种生物的基因组进行了编辑。由此产生的基因编辑动物的建系与鉴定是随之而来较为繁琐的工作。单导向RNA(single-guide RNA,sgRNA)靶序列的设计和确定不仅影响后续靶向基因组的效率,还可作为优化鉴定、筛选方法的参考。本研究在选取sgRNA靶序列时,不仅依据软件的评分,还分析了sgRNA靶序列是否含有酶切位点,以便对后续纯合子/杂合子进行鉴定。结果显示,以特异引物扩增的野生型小鼠Chrm3基因片段可被限制性内切酶BanⅡ切为两个片段;而纯合子小鼠"丢失"该酶切位点,其PCR产物不能被切开;杂合子小鼠PCR产物被不完全切开,凝胶电泳结果可见三条带。本研究结果提示该策略可有效简化基因编辑动物建系鉴定工作,提高鉴定效率及改善阳性动物辨识效果。CRISPR / Cas9-based gene editing technology has developed rapidly in recent years.Establishing lines of genomic modified animals requires the verification of founders. For mice generated using CRISPR / Cas9 system,this tedious procedure is also needed. By incorporating proper restriction sites in the design of single-guide RNA（ sgRNA） can be helpful for the identification of establishment lines. To better verify homozygous and heterozygous mice,we selected sgRNA sequences by scores from predicting software and presence of specific restriction sites. We produced a genetically modified Chrm3 mice by cytoplasm micro-injection. The PCR products from Chrm3 mice DNA were digested by BanⅡ.The results showed that the sample from WT mice had two bands after restriction; homozygous Chrm3 mice bearing no restriction sites showed only one band,and the heterozygotes gave three bands. Our attempted strategy,to some extent,simplified the line establishment verification and homozygous /heterozygote identification processes.

关 键 词：CRISPR/Cas9 毒蕈碱型乙酰胆碱受体M3亚型 遗传改变小鼠 纯合子 

分 类 号：Q78[生物学—分子生物学]