原位染色检测慢病毒载体转录通读方法的建立  

The Establishment of the Methodology used for In Situ Detecting Lentiviral Vector Transcriptional Read-through

作　　者：高越[1,2,3] 檀硕 任兆瑞[1,2,3,5] 张敬之[1,2,3,4] 

机构地区：[1]上海交通大学附属儿童医院 [2]上海市儿童医院 [3]上海交通大学医学遗传研究所,上海200040 [4]卫生部医学胚胎分子生物学重点实验室暨上海市胚胎与生殖工程重点实验室,上海200040 [5]生物治疗协同创新中心四川大学华西医院,成都610041

出　　处：《中国生物工程杂志》2015年第5期55-60,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(81271690)

摘　　要：慢病毒载体作为基因导入系统已被多次应用于基因治疗试验当中,近年来受到了广泛关注。转录通读是慢病毒载体应用过程中存在的潜在不安全因素之一。为了进一步完善慢病毒载体转录通读的检测方法,使检测结果更加直观准确,在已建立的在转录水平检测其转录通读的基础上,进一步模拟病毒载体整合入基因组的状态,于原病毒载体3'-LTR后插入经密码子优化的Lac Z报告基因。经转染细胞后进行原位染色,发生通读现象的细胞会被染成蓝色,从而在蛋白质水平直观地体现慢病毒载体的通读情况,说明所建立的方法是准确可行的。所得结论与q RTPCR方法在转录水平检测的结果是平行相一致。原位染色检测法的建立为慢病毒载体转录通读的检测、提高慢病毒载体生物安全性的研究提供了技术支持。As a gene delivery system, Lentiviral vector （LVV） has been being therapy trials, and drawn large attention in recent years. Yet, transcriptional read- increasingly used in gene through （TRT） is one of potential risks in its application. For the purpose of improving the detection methods of TRT, and making the results intuitive and accurate, a codon optimized reporter gene, LacZ, was placed downstream to the 3＇- LTR of the proviral vector to simulate its state in chromosome. Upon the transfection followed with LacZ staining, the cells with TRT phenomenon would be stained blue, and the TRT rate would therefore be visualized at the translational level. Correlating well with the outcomes of qRT-PCR, the established methodology could reflect the feature of TRT, which paves a way for improving LVV biosafetv for clinical applications

关 键 词：慢病毒载体 转录通读 原位染色 原病毒载体 

分 类 号：Q78[生物学—分子生物学]