Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsWe 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.
Highlights
The transposon-based gene delivery technique is emerging as a method of choice for gene therapy
To optimize the qPCR-TI method, we began with clones of HEK-293 cells with Sleeping Beauty (SB) transposons carrying two transcription units expressing green fluorescent protein (GFP) and the puromycin-resistance gene, which are both under the control of the CAG promoter (Figure 1A)
Specific TaqMan® (Applied Biosystems, Foster City, CA, USA) assays were designed for the two inverted repeat-direct repeat (IRDR) motifs of the SB transposon and for the GFP sequence (Figure 1A)
Summary
The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. A novel hyperactive version of the Various technical methods have been developed to determine transgene copy numbers after gene delivery, including Southern blotting and the specific PCR-based transposon display method [14,15]. In most cases, these are performed using radioactively labeled probes; fluorescent labeling can be used, its threshold detection levels are generally lower. Depending on the transgene used, other techniques such as in situ hybridization quantification of fluorescent marker proteins such as green fluorescent protein (GFP) can be employed [16] These methods are widely accepted and used, they are usually laborious and require specific chemicals and equipment. These detection methods are often limited to the measurement of a specific transgene, and lengthy pilot experiments are often required to determine the exact measurements needed to accurately quantify a newly arising gene of interest within a particular delivery system [17,18,19]
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