慢病毒载体转录通读率检测方法的建立

Establishment of the methodology for quantifying lentiviral vector transcriptional read-through rate

摘要:

慢病毒载体作为一种有效的生物研究和基因治疗载体，近年来正受到广泛关注，而转录通读现象则是限制其被使用的主要生物安全性瓶颈之一。为了评估慢病毒载体在整合入染色体后的转录通读的实际情况，需要建立一种有效的检测转录通读率的方法。文中运用分子生物学等手段，将相关质粒瞬时转染入293T细胞，模拟野生型和自灭活型慢病毒载体整合入染色体后的情况，利用实时荧光定量PCR分别定量转录通读和总转录本在慢病毒载体上的特征序列，并计算出转录通读率；同时使用流式细胞计数等技术，检测转录通读后的GFP蛋白产物。两种检测结果均与理论相符，即自灭活型载体具有更高的转录通读率。说明文中所建立的方法有效可靠，为进一步评估和降低慢病毒载体的转录通读率，提高慢病毒载体的生物安全性的研究提供技术支持

Abstract:

As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand?the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of “proviral vectors” in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of?LV