使用TALENs技术构建丁酰胆碱酯酶敲除小鼠模型研究  

Study on create butyrylcholinesterase knock out mouse model by TALENS

作　　者：胡婷婷[1] 赵海山 张昊[1] 谭文 HU Tingting ZHAO Haishan ZHANG Hao TAN Wen(School of Bioscience and Bioengineering, Pre - Incubator for Innovative Drugs and Medicine Center, South China University of Technology, Guangzhou 510006, China Institute of Biomedical ＆ Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China)

机构地区：[1]华南理工大学生物科学与工程学院/生物医药前孵化器研究中心,广州510006 [2]广东工业大学生物医药研究院,广州510006

出　　处：《黑龙江畜牧兽医》2017年第10期36-38,45,288-290,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：广东省科技厅省重大专项合作项目(2015B010109004)

摘　　要：为了使用转录激活因子样效应物核酸酶(TALENs)技术构建丁酰胆碱酯酶(BChE)敲除小鼠模型,试验设计多对TALENs组合,合成相应的载体,并通过3T3细胞系对这些载体的活性进行检测,选择出活性最高的组合进行体外转录获得TALENs mRNA,并注射入小鼠受精卵中,对子代小鼠进行基因测序并筛选,最终获得BChE基因敲除小鼠。结果表明:针对4个基因位点设计并构建了30对TALENs组合,选择最优3L3-3R1组合进行体外转录并注射入小鼠受精卵,获得4只携带突变的F0代嵌合体小鼠;通过杂交及筛选,最终获得4只携带移码突变的F1代小鼠,可用于交配繁殖。说明通过TALENs技术可以稳定可靠地制备BChE基因敲除小鼠,并用于后续BChE功能的研究。The aim of the present study was to construct butyrylcholinesterase （BChE） knockout mice model by using the transcriptional activa- tor -like effector nuclease （TALENs） technique. Many pairs of TALENs were designed in this experiment, and the corresponding vectors were synthesized. Then the activity of these vectors was detected in the 3T3 cell line. The most active TALENs were chose and transcribed in vitro to obtain TALENs mRNA. Then the mRNA was injected into the mouse fertilized eggs. The BChE knockout mice were eventually obtained by gene sequencing and screening of offspring mice. The results showed that 30 pairs of TALENs were designed and constructed for four gene loci. The best combination 3L3 -3R1 was chose and transcribed in vitro. Then four mutant F0 chimeric mice were obtained after the mRNA was injected into the mouse fertilized eggs in vitro. Finally, four FI - generation mice carrying the frameshift mutations that could be used for mating and breeding were obtained by hybridization and screening. The results showed that the BChE knockout mice could be stably and reliably obtained by TALENs technique. The mice could also be used for subsequent BChE function studies.

关 键 词：丁酰胆碱酯酶(BChE) 转录激活因子样效应物核酸酶(TALENs) 载体构建 小鼠 基因敲 除 纯合子 

分 类 号：Q785[生物学—分子生物学]