Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

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.Transposon-based systems have become the method of choice for gene delivery, and their applications as potential genetic vehicles are receiving great interest [1-3]. In recent years, the Sleeping Beauty (SB) transposon has been emerging as the most favorable delivery system, because of its random integration profile and the lack of similar transposon-like elements in the human genome, which significantly minimizes the risk often represented by viral-based methods [4-6]. Owing to its advantageous characteristics, SB is the first transposon-based system to be used in a clinical trial for a hematologic malignancy [7]. Recently, a novel hyperactive version of the originally reconstituted SB transposase was developed [8], which, apart from making the system more favorable than other widely used non-viral methods, further substantiates its applicability as a mutagenic tool to perform genetic analyses, similar to the transposon-based systems in D. melanogaster a