原位染色检测慢病毒载体转录通读方法的建立

发布时间：2018-03-05 16:45

本文选题：慢病毒载体　切入点：转录通读　出处：《中国生物工程杂志》2015年05期 　论文类型：期刊论文

【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。
[Abstract]:Lentiviral vectors as gene delivery system has been applied in gene therapy trials, in recent years has been widely concerned. Transcription readthrough is one of the potential risk factors of lentiviral vectors in the application process. In order to further improve the lentiviral vector transcription readthrough detection method, which makes the results more intuitive and accurate, has been based on the detection of the transcriptional readthrough at the transcriptional level, further simulation of viral vectors integrated into the genome of the state, from the original virus vector 3'-LTR after insertion of the codon optimized Lac Z reporter gene. The transfected cells after in situ staining, the cells will be read through the phenomenon of occurrence of blue, read to reflect lentiviral vectors at the protein level, shows that the method is accurate and feasible. The results with the Q RTPCR method at the level of transcription was flat The establishment of in situ staining assay provides a technical support for the detection of lentiviral vectors' transcriptional reading and improving the biological safety of lentiviral vectors.

【作者单位】：上海交通大学附属儿童医院;上海市儿童医院;上海交通大学医学遗传研究所;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;生物治疗协同创新中心四川大学华西医院;
【基金】：国家自然科学基金资助项目(81271690)
【分类号】：R373;Q78

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