CAJ | 학술논문

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。
보고재체계통인구건쾌첩、개조간단、조작용역、경제유효，병차능통과개도사선핵산매양성세포부집기인조수식적양성세포극륭，이성위특이성핵산매활성검측적중요수단。기우비동원말단련접(Non-homologous end joining，NHEJ)수복궤제적보고계통재인입DNA쌍련단렬(Double strand breaks，DSBs)후，경과우화최고야지유2/3적개솔실현보고기인적수복；이단련퇴화(Single strand annealing，SSA)수복궤제재인입DSBs후，이론상가이실현보고기인100%적수복，구유경고적령민도，유리우활성교저적특이성핵산매활성적검측，위기인조수식연구중특이성핵산매활성적검측제공료유효적수단。본연구설계병구건료3투기우 SSA 수복궤제적쌍형광보고재체계통，병응용mRFP-eGFP계통검측료3대ZFNs적유효활성，기활성검측결과분별위8.9%、9.3%화5.0%，해연구위핵산매활성적검측제공료유효적수단。
Reporter vector system has become an important method for measuring activity of specific nucleas-es 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 in-troducing 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 de-tection 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.