Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary.
Mob DNA. 2011 Mar 3;2(1):5. doi: 10.1186/1759-8753-2-5.
The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo.
We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle.
We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.
转座子为基础的基因传递技术正成为基因治疗的首选方法之一。睡眠美人（SB）系统因其效率和随机整合特性已成为最受欢迎的方法之一。传递的转基因的拷贝数确定是一项关键任务，但缺乏通用的测量方法。在本文中，我们表明，一种基于实时定量 PCR 的、与转基因无关的（qPCR-TI）方法能够确定 SB 转座子拷贝数，而不受遗传货物的影响。
我们设计了一种特定的 PCR 检测来扩增 SB 的左反向重复-重复序列区，并与单拷贝对照基因 RPPH1 和具有已知拷贝数的参考基因组 DNA 一起使用。qPCR-TI 方法允许在各种细胞类型中快速准确地确定 SB 转座子拷贝数，包括人胚胎干细胞。我们还发现，这种灵敏、快速、高度可重复和非放射性的方法与广泛使用的印迹技术或转座子显示方法一样准确可靠。由于该检测针对转座子的反向重复区，因此可用于任何以 SB 转座子作为遗传载体的系统中。
我们开发了一种与转基因无关的方法来确定睡眠美人转座子系统传递的转基因的拷贝数。该技术基于定量实时 PCR 检测方法，提供了一种灵敏、非放射性、快速和准确的方法，具有用于基因治疗的潜力。
