基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用

doi:10.16288/j.yczz.15-246

遗传 ›› 2015, Vol. 37 ›› Issue (10): 1053-1060.doi: 10.16288/j.yczz.15-246

基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用

韩芙蓉¹, 王令^1,2, 徐坤¹, 张智英¹, 王昕¹

1. 西北农林科技大学动物科技学院,杨凌 712100;
2. 陕西理工学院生物科学与工程学院,汉中 723000

收稿日期:2015-06-01 出版日期:2015-10-20 发布日期:2015-10-20
通讯作者: 王昕,博士,教授,研究方向：生物技术与家畜育种。E-mail: wxwza@126.com
作者简介:韩芙蓉,硕士研究生,专业方向：动物遗传育种与繁殖。E-mail: hanfr611miffy@sina.com
基金资助:
陕西省科技攻关项目(编号：2014K02-07-01)资助

Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism

Furong Han¹， Ling Wang^1，2， Kun Xu¹， Zhiying Zhang¹， Xin Wang¹

1. College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China;
2. School of Biological Science and Engineering, Shaanxi University of technology, Hanzhong 723000, China

Received:2015-06-01 Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： 报告载体系统因构建快捷、改造简单、操作容易、经济有效,并且能通过介导筛选核酸酶阳性细胞富集基因组修饰的阳性细胞克隆,而成为特异性核酸酶活性检测的重要手段。基于非同源末端连接(Non-homologous end joining,NHEJ)修复机制的报告系统在引入DNA双链断裂(Double strand breaks,DSBs)后,经过优化最高也只有2/3的概率实现报告基因的修复;而单链退火(Single strand annealing,SSA)修复机制在引入DSBs后,理论上可以实现报告基因100%的修复,具有更高的灵敏度,有利于活性较低的特异性核酸酶活性的检测,为基因组修饰研究中特异性核酸酶活性的检测提供了有效的手段。本研究设计并构建了3套基于SSA修复机制的双荧光报告载体系统,并应用mRFP-eGFP系统检测了3对ZFNs的有效活性,其活性检测结果分别为8.9%、9.3%和5.0%,该研究为核酸酶活性的检测提供了有效的手段。

关键词: SSA, 双荧光报告载体系统, 特异性核酸酶

Abstract: Reporter vector system has become an important method for measuring activity of specific nucleases because of its fast construction, simple modification, easy operation, economic effectiveness as well as its role in enriching positive cells with genomic modification through mediating screen of specific nucleases positive cells. After introducing double strand breaks (DSBs), a reporter system based on non-homologous end joining (NHEJ)-mediated repair can only repair maximally two thirds of reporter genes after optimization, while single strand annealing (SSA)-mediated repair can repair all reporter genes theoretically which has higher sensitivity and facilitates the detection of specific nuclease with low activity and provides an effective way to detect specific nuclease activity in genome modification studies. In this study, we designed and constructed three sets of dual-fluorescence reporter systems based on SSA repair mechanism and applied the mRFP-eGFP system in measuring the effective activity of three pairs of ZFNs, which was 8.9%, 9.3% and 5.0%, respectively. Our study provides an effective way to detect the activity of nucleases.

Key words: SSA, dual-fluorescence reporter system, specific nucleases

韩芙蓉, 王令, 徐坤, 张智英, 王昕. 基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用[J]. 遗传, 2015, 37(10): 1053-1060.

Furong Han， Ling Wang， Kun Xu， Zhiying Zhang， Xin Wang. Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism[J]. HEREDITAS(Beijing), 2015, 37(10): 1053-1060.

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基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用

Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism

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