Reliable transgene-independent method for determining <it>Sleeping Beauty </it>transposon copy numbers

• Main Authors: Kolacsek Orsolya, Krízsik Virág, Schamberger Anita, Erdei Zsuzsa, Apáti Ágota, Várady György, Mátés Lajos, Izsvák Zsuzsanna, Ivics Zoltán, Sarkadi Balázs, Orbán Tamás I
• Format: Article
• Language: English
• Published: BMC 2011-03-01
• Series: Mobile DNA
• Online Access: http://www.mobilednajournal.com/content/2/1/5

<p>Abstract</p> <p>Background</p> <p>The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The <it>Sleeping Beauty </it>(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.</p> <p>Results</p> <p>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 <it>RPPH1 </it>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.</p> <p>Conclusions</p> <p>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.</p>