Open Access Methodology

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

Orsolya Kolacsek1, Virág Krízsik1, Anita Schamberger1, Zsuzsa Erdei1, Ágota Apáti1, György Várady1, Lajos Mátés2, Zsuzsanna Izsvák23, Zoltán Ivics23, Balázs Sarkadi1 and Tamás I Orbán1*

Author Affiliations

1 Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary

2 Mobile DNA Group, Max-Delbrück Center for Molecular Medicine, Berlin, Germany

3 Department of Human Genetics, University of Debrecen, Debrecen, Hungary

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Mobile DNA 2011, 2:5  doi:10.1186/1759-8753-2-5

Published: 3 March 2011

Abstract

Background

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.

Results

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.

Conclusions

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.