Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers METHODOLOGY Open Access 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 Izsvák2,3, Zoltán Ivics2,3, Balázs Sarkadi1, Tamás I Orbán1* 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. Background Transposon-based systems have become the method of choice for gene delivery, and their applications as poten- tial 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 malig- nancy [7]. Recently, a novel hyperactive version of the originally reconstituted SB transposase was developed [8], which, apart from making the system more favor- able than other widely used non-viral methods, further substantiates its applicability as a mutagenic tool to per- form genetic analyses, similar to the transposon-based systems in D. melanogaster and C. elegans [9,10]. Although already possessing clear advantages, rigorous characterization of the SB system still remains to be car- ried out to set up standard methods concerning its applicability. One of the important issues in setting up gene-therapy guidelines or genome-wide mutagenesis protocols is that of copy-number determination in stable clones [11-13]. 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 * Correspondence: orbant@biomembrane.hu 1Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary Full list of author information is available at the end of the article Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 © 2011 Kolacsek et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mailto:orbant@biomembrane.hu http://creativecommons.org/licenses/by/2.0 are performed using radioactively labeled probes; although fluorescent labeling can also be used, its threshold detection levels are generally lower. Depend- ing on the transgene used, other techniques such as in situ hybridization quantification of fluorescent marker proteins such as green fluorescent protein (GFP) can also be employed [16]. Although these methods are widely accepted and used, they are usually laborious and require specific chemicals and equipment. In addition, these detection methods are often limited to the mea- surement 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 sys- tem [17-19]. During this study, we aimed to develop an accurate method for quantifying SB transposon copy numbers, independent of the transgene sequence. We term this the real-time quantitative PCR-based, transgene-inde- pendent (qPCR-TI) method. It can be used for any SB- based gene delivery experiments without a priori opti- mization of the protocol. To establish this method, we used specific probe sets designed for the left and right inverted repeat-direct repeat (IRDR) regions, which are the recognition motifs of the transposase and therefore required for any SB transposition reaction [20]. As an internal control for normalization, a probe for the RPPH1 gene, the H1 RNA subunit of the RNaseP enzyme complex, was used. This gene is a widely accepted one-copy gene of the haploid human genome [21]. Comparing this system with the radioactive transposon display and Southern/ dot blotting techniques, we provide evidence that using the IRDR-L specific probe set in comparative 2-ΔΔCt measurements can reliably and accurately quantify SB transposon copy numbers in various cell lines, regardless of the transgene used. Apart from being sensitive, accu- rate and rapid, this real-time PCR-based quantification method also offers a powerful non-radioactive technique as an alternative against other standard methods. Results and Discussion The exact and rapid quantification of transgene copy numbers is often required for gene-delivery experiments. As we generally use the SB transposon system in our laboratory, we aimed to develop a real-time PCR-based technique that would be transgene-independent, specific for the transposon regions and therefore widely applic- able. To optimize the qPCR-TI method, we began with clones of HEK-293 cells with SB transposons carrying two transcription units expressing GFP and the puromy- cin-resistance gene, which are both under the control of the CAG promoter (Figure 1A). This transgene setup allowed generation of clones with various copy numbers by either fluorescence-activated cell sorting (FACS) or antibiotic selection. Specific TaqMan® (Applied Biosys- tems, Foster City, CA, USA) assays were designed for the two IRDR motifs of the SB transposon and for the GFP sequence (Figure 1A). The widely applicable SB transposon version used throughout this study has two asymmetric IRDR regions (’left’ and ‘right’ [22]). In most transposon flanking sequences, the two IRDR regions are repeat-rich DNA sequences, which makes PCR pri- mer design relatively difficult. Moreover, the left and the right IRDRs are very similar to each other, which further increases the difficulty of designing specific assays for them. Nevertheless, we could still develop specific assays for each; neither of the IRDR-L nor the IRDR-R probe set gave signals in the exclusive presence of the other template (data not shown). As the first (and simplest) approach, absolute quantifi- cation of DNA samples was performed using plasmid dilution series complemented with transposon-free non- specific genomic (g)DNA. However, the difficulties of determining the exact nucleic-acid concentration of very dilute samples and the differences in purity between sam- ples made it necessary to abandon absolute quantifica- tion, and to include an internal copy control to overcome these problems with relative quantification. The RPPH1 gene, the H1 RNA subunit of the RNaseP enzyme com- plex, was chosen as this is a widely-accepted one copy gene of the haploid human genome [21] (http://www. ncbi.nlm.nih.gov/ieb/research/acembly/index.html). However, the assay efficiency for the IRDR-R region dif- fered significantly from that of the others, including the RPPH1 endogenous control assay. Various conditions for the IRDR-R set were tried, and although template con- centration seemed to be a crucial factor, the widely accepted template range of 10 to 40 ng still produced efficiency values that were significantly lower than those of the other assays (<90%) (Figure 1B). Sequence con- straints originating from the similarity to IRDR-L hin- dered us designing other specific assays with different combinations of primers and probes in this short (228 bp) and repeat-rich region. Therefore, if this assay were to be included for measurement, the relative standard curve method would be the only acceptable quantifica- tion method, as it is the most suitable to compare reac- tions with suboptimal PCR efficiency. Apart from the setting up of standard curves (for both the transposon- specific assays and the RPPH1 endogenous control), rela- tive quantification also requires the use of a calibrator (a reference sample with a known copy number, preferably ‘1’) to ensure the precision of quantification. In the search for a potential calibrator sample, gener- ated clones were screened by FACS for the lowest possi- ble GFP signal, assuming that clones with one copy number should be among those samples (the signal Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 2 of 8 http://www.ncbi.nlm.nih.gov/ieb/research/acembly/index.html http://www.ncbi.nlm.nih.gov/ieb/research/acembly/index.html could also vary because of positional effects of different integration sites). Although the CAG promoter we used is known to be less prone to silencing [23-25], we had to make sure the lowest fluorescent signals were also associated with the lowest real-time signals when normalized to the RPPH1 level, in order to exclude the potential presence of silenced copies. Using the GFP TaqMan® assay, several clones with one integrated transposon copy and numerous others with three or four copies were found (Figure 2A,B). Using the IRDR-L Figure 1 Real-time PCR assay designed for different transposon and transgene regions. (A) Structure of the used SB transposons with asymmetric IRDRs [22]. For each construct, the TaqMan® assays (TQ) used for copy-number determination are indicated. Sequences are not drawn to scale. IRDR-L/-R = inverted repeat-direct repeat left/right regions; pA = SV40 polyadenylation signals. (B) Efficiencies of the real-time assays determined by standard curves. For all assays, a dilution series was prepared from pooled genomic DNA samples from clones containing integrated transposon 1. The efficiency of the IRDR-R TaqMan® assay was notably lower than that of the others (<90%). Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 3 of 8 set, very similar copy numbers could be calculated using the relative standard curve method (Figure 2C), whereas the IRDR-R TaqMan® set gave unreliable results, mainly due to the problems discussed earlier (Additional file 1). Because the assays for RPPH1, GFP and the transposon IRDR-L had very similar efficiency values (Figure 1B), we also tried another approach, calculating the copy numbers in the examined clones by the comparative Ct (2-ΔΔCt) method in the same experiments. The results based on GFP or IRDR-L were in agreement with each other and with the results of the relative standard curve method. Moreover, technical errors could be further decreased by using a pool of gDNA samples with known copy number as a reference. We therefore concluded that once we left out the specific but less effi- cient assay for the IRDR-R region, the comparative Ct method could be used for reliable and precise transpo- son copy-number determination using the IRDR-L Taq- Man® assay. Abandoning the relative standard curve method also allowed inclusion of more samples in one reaction plate, as no more dilution series with several parallels were required. To test the qPCR-TI method on other samples, we exam- ined clones of the HUES9 human embryonic stem cell line expressing the GFP-tagged ABCG2 transporter [26] gener- ated by the SB transposon system. Again, the GFP and the IRDR-L TaqMan® assays could be compared with each other (Figure 1A, transposon 2). As a general assay setup, Figure 2 Comparing copy-number determination by green fluorescent protein (GFP) or transposon-specific real-time PCR. (A) Fluorescence-activated cell sorting (FACS) analysis of different HEK-293 derived clones expressing GFP. Higher fluorescent intensities indicate higher copy numbers, although signals can vary because of integration position effects and/or transgene silencing. The control sample shows the autofluorescence detected in non-transfected HEK-293 cells. (B) Copy numbers determined by transgene (GFP) specific real-time PCR assay normalized to the level of one copy control RPPH1; clones analyzed by FACS (A) and other clones established subsequently were examined. Various clones with low GFP expression level were determined to have one integrated transposon copy, whereas the majority with higher GFP fluorescence was found to have four transposon copies. In the case of clone 5.a, further analysis revealed that it was not a clone but rather a mixture of clones with an average copy number around 4.5. (C) Comparison of two techniques. The copy values determined by the transgene independent TaqMan® assay for the IRDR-L sequence correlated well with the GFP-based copy numbers. Clone 2.r originated from random integration, so the transposon repeat sequence might not be intact, and the partial presence of IRDR-L could result in a lower signal, therefore this clone was not included among the controls for later experiments. a = clones obtained from active transposition; r = clones obtained from random integration (from transfection with the mutant transposase). For copy numbers, values are means ± SEM of at least three independent measurements. Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 4 of 8 the RPPH1 control and reference samples (pool of clones with known copy numbers) were used. As shown in Figure 3, the 2-ΔΔCt method produced the same copy num- bers, using either of the probe sets. These experiments therefore supported the use of the IRDR-L repeat specific assay for transposon copy-number determination, as it gave the same results as the assay specific for the carried internal transgene. To compare our transgene-independent quantification approach with other techniques, we measured copy numbers of clones generated from HeLa cells by trans- posons containing a neomycin-resistance (neoR) gene (Figure 1A, transposon 3). Such clones were ideal for comparison because of the different transgene sequences and because their copy numbers were also determined by the Southern/dot blotting techniques or the transpo- son display method [5]. Several clones were tested, and the qPCR-TI method gave the same copy numbers as determined by the other radioactive methods (Table 1). For higher (>5) copy-number clones, the qPCR-TI method was also reasonably accurate, with occasional low relative-error margins (≤9%). The slight differences in some cases could be due to the inaccuracy of the standard methods for this range [14,15]. In addition, it has been suggested that precise values of very high copy numbers are more reliably measured by dot blot rather than transposon display methods. We found that the copy number of clone 4 determined by the dot-blot technique correlated well with data produced by the qPCR-TI. For low copy-number clones, only one clone (2/2 of neoR; see Table 1) did not give identical results with the different techniques. A difference of one copy number here clearly represents a higher percentage error margin, but this error might be related to the dif- ference in integration sites in that particular clone (see discussion below). Taken together, the results of the neoR transposon clones indicated that the qPCR-TI technique is just as sensitive and accurate as the other widely used methods. A further proof of principle was given by the determi- nation of the transposon copy numbers in HUES9 clones previously generated using another sequentially distinct transgene. In those experiments, the amaxaGFP (a special fluorescent protein from a Pontellina copepod species, http://www.lonzabio.com) was carried by the transposon to generate clones of an embryonic stem-cell line, and the transposon integration sites were deter- mined by the splinkerette PCR and the inverse PCR methods [27]. Based on these integration assays, copy numbers were estimated to be one to six in various clones, although all integrated copies may not be reliably detected by these methods because of the different flanking genomic sequences. When using the qPCR-TI method for several clones using the IRDR-L assay, the measured transposon copies were almost always the same as those previously claimed on the basis of the dif- ferent proven integration sites (Table 1). One exception here was clone B1, where qPCR-TI gave a result one copy higher, similarly to the 2/2 neoR clone. A differ- ence of one copy number here again undoubtedly repre- sents a higher discrepancy with higher percentage error margin. However, because all the other low copy-num- ber clones gave identical results with the various techni- ques, the two outliers might represent the lower sensitivity of the standard methods due to the depen- dence of transgene-integration sites [15]. These compar- isons lead us to the conclusion that the qPCR-TI method provides reliable results for different SB trans- poson constructs, thereby being a consistent transgene- independent copy-number quantification method. Using the experiments described above, the newly developed transgene-independent method for determin- ing SB transposon copy numbers was validated: (i) it provided the same results as the assays specific for the carried transgene sequence and (ii) it could also reliably replace widely used standard radioactive techniques. The TaqMan® assay designed for the IRDR-L region of the transposon provides the basis for transgene indepen- dence as it is present in all SB constructs. In fact, ‘sym- metric’ SB transposons with two IRDR-L (but not two IRDR-R) flanking sequences are functional [28], and the qPCR-TI method is also applicable to such constructs (with an obvious correction factor of 0.5). We found evi- dence that the PCR efficiency of this probe set is similar to the RPPH1 single-copy control, so reliable quantifica- tion can be performed using the comparative 2-ΔΔCt method. To ensure precise and rapid quantification, Figure 3 Copy-number determinations of green fluorescent protein (GFP)-ABCG2 expressing HUES9 clones. The sample ‘pool’ indicates the equimolar mixture of gDNA samples from the first four single-copy clones on Figure 2C. Later examination of the G2C3 line indicates that it is not derived from a clone but rather from a mixture of cells with five and six transposon copies. Values are means ± SEM of at least three independent measurements. Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 5 of 8 http://www.lonzabio.com reference samples (calibrators) with known copy num- bers are also included, preferably a pool of gDNAs from different clones, to minimize discrepancies resulting from different transgenic sampling techniques and puri- ties. The method could also be extended to other non- human gDNA samples; however, a suitable and validated single-copy reference gene control must always be used. Another technical point that should be considered is the transposition-independent, random integration of the transgene. Because this is a stochastic process, it could possibly lead to the integration of the carried transcription unit without the transposon IRDR sequences. In such cases, the qPCR-TI method clearly underestimates transgene copy numbers, as it only detects copies resulted from bona fide transposition. As a general rule, we always include control experiments with gene delivery using the mutant transposase to esti- mate the level of random integration [20]. According to previous experiments, this phenomenon is generally very rare when using the new hyperactive SB100x transpo- sase, but its extent can vary between different cell lines. Nevertheless, if such random background integration increases significantly, it may be necessary to measure the copy numbers of the transgene itself in the samples generated with the active transposase. Conclusions We have developed a sensitive and reliable real-time PCR-based (qPCR-TI) method for measuring SB transpo- son copy numbers. When compared with widely used standard methods, such as various blotting techniques or transposon display, it proved to be just as accurate as those other methods, while also offering a faster and non-radioactive method. However, the real advantage of this method is the transgene independence, which makes it applicable for any scientists working with Sleeping Beauty transposon constructs. Therefore, we believe that qPCR-TI could become the method of choice for gene therapy and general gene-delivery applications. Methods Cell-culture maintenance and creation of clones Human embryonic kidney cells (HEK-293) were cul- tured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% L-glutamine and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Transfected cell populations were first enriched for transgene expression by flow cytometry (see below). Subsequently, cell clones were created by serial dilutions in 96-well plates. Selected clones were further analyzed by flow cytometry and harvested for Table 1 Comparing the qPCR-TI method with other standard techniques Clone name Methods Copy numbers By standard methods By qPCR-TIa Transposons carrying the neomycin resistance gene 2/1 Transposon display/Southern blotting 8 to 10 8 2/2 Transposon display/Southern blotting 3 4 2/3 Transposon display/Southern blotting 10 to 12 10 2/9 Transposon display/Southern blotting 1 1 1 Transposon display/Southern blotting 12 to 13 13 4 Dot blot 52 50 5 Transposon display/Southern blotting 15 15 6 Transposon display/Southern blotting 12 11 7 Transposon display/Southern blotting 1 1 8 Transposon display/Southern blotting 2 2 9 Transposon display/Southern blotting 1 1 Transposons carrying the amaxaGFP transgene A3 Splinkerette PCR/inverse PCR 2 2 A4 Splinkerette PCR/inverse PCR 4 4 A5 Splinkerette PCR/inverse PCR 4 4.5b A6 Splinkerette PCR/inverse PCR 2 2 B1 Splinkerette PCR/inverse PCR 1 2 B3 Splinkerette PCR/inverse PCR 3 3 B5 Splinkerette PCR/inverse PCR 2 2 aQuantitative PCR, transgene independent. bFor sample A5 from the amaxa green fluorescent clones, real-time PCR measurement indicated that it is more likely to be a mixed population of cells rather than a single clone. Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 6 of 8 genomic DNA isolation (see below). The HUES9 embryonic stem-cell line (originally provided by Dr. Douglas Melton, Harvard University, USA) was main- tained essentially as described previously [29], using cells from passage 35. To create transgene-expressing HUES9 clones, we used our previously developed method for human embryonic stem-cell lines [27]. Transfection and transposition HEK-293 and HUES9 cells were transfected using a transfection reagent (FuGENE® 6; Roche Applied Science, Rotkreuz, Switzerland) in accordance with the manufacturer’s instructions. The transfection mix con- tained 1 μg of a given transposon plasmid (Figure 1A) and 100 ng of the hyperactive SB100x Sleeping Beauty transposase, in a 10:1 ratio to minimize the overproduc- tion inhibition phenomenon [5,8]. To visualize the ran- dom integration background, a control transfection with the inactive DDE motif mutant of the transposase was carried out, using the same experimental setup [20]. Flow cytometry GFP-expressing cells were analyzed by a flow cytometer (FACSCalibur; Becton-Dickinson, San Jose, CA, USA) with Cellquest-Pro analysis software (Becton-Dickinson). Mock-transfected cells were used as labeling controls, and propidium iodide or 7-aminoactinomycin D staining was used to exclude non-viable cells. To select and clone cells expressing GFP, a fluorescence based cell sorter (FACSAria High Speed Cell Sorter; Becton-Dick- inson) was used in accordance with the manufacturer’s instructions. Genomic DNA isolation, transposon display and Southern/dot blotting After treatment with trypsin, cells were separated by centrifugation and washed with 1 × phosphate-buffered saline. After careful removal of the liquid supernatant, the dry cell pellets were stored at -80°C until further processing. Genomic DNAs were isolated from the cells by standard phenol-chloroform extraction after cell lysis and proteinase K digestion. DNA samples were quanti- fied with a spectrophotometer (GeneQuant II; Pharma- cia Biotech, Piscataway, NJ, USA). Transposon display and Southern-/dot-blotting techniques were performed essentially as described previously [5,14]. Quantitative real-time PCR Reactions were performed on a real-time PCR platform (StepOne™ or StepOnePlus™; Applied Biosystems, Fos- ter City, CA, USA) in accordance with the manufac- turer’s instructions. The gDNA samples (30 ng each) were run in triplicate, in singleplex reactions with a final volume of 20 μl using TaqMan® chemistry. All primers and probes were designed by Primer Express software (version 3.0; Applied Biosystems), and probes were labeled with 5’-FAM and 3’-nonfluorescent (minor groove binding) quencher molecules. Sequences for the TaqMan® assays are given in Table 2. Final concentra- tions of primers and probes were 250 and 900 nM, respectively. Data were analyzed by StepOne software (version 2.1; Applied Biosystems). Additional material Additional file 1: Supplementary Figure 1: Comparison of the IRDR- R assay with the GFP specific real-time PCR method. Selected HEK- 293 clones were examined for transposon copy numbers in parallel by the accepted green fluorescent protein (GFP) specific assay and the assay specific for Sleeping Beauty (SB) inverse repeat-direct repeat, right (IRDR)- R. In contrast to the IRDR, left (IRDR-L) real-time assay, the IRDR-R specific assay failed to reproduce previously determined copy numbers consistently (see Figure 2C). For this particular experiment, 30 ng genomic (g)DNA was used for the reaction. Although different starting gDNA concentrations (higher than the recommended range of 10 to 40 ng) improved the reproducibility of the IRDR-R assay, it still did not reach the reliability level of the GFP or the IRDR-L assays. Acknowledgements We thank Dr Douglas Melton for the gift of the HUES9 cell line. T I O is a recipient of the János Bolyai Scholarship from the Hungarian Academy of Sciences. This work was supported by grants from OTKA (NK72057), ETT (213-09), ES2Heart Jedlik (OM00203/2007), STEMKILL Jedlik (OM00108/2008) and National Development Agency grant KMOP-1.1.2-07/1-2008-0003. Author details 1Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, Budapest, Hungary. 2Mobile DNA Group, Max-Delbrück Center for Molecular Medicine, Berlin, Table 2 Primers and probes used for quantitative real-time PCR Primer/probe name Sequence 5’®3’ RPPH1 Forward AGCTGAGTGCGTCCTGTCACT Reverse TCTGGCCCTAGTCTCAGACCTT Probe CACTCCCATGTCCC GFP Forward GAGCGCACCATCTTCTTCAAG Reverse TGTCGCCCTCGAACTTCAC Probe ACGACGGCAACTACA IRDR-L Forward CTCGTTTTTCAACTACTCCACAAATTTCT Reverse GTGTCATGCACAAAGTAGATGTCCTA Probe CTGACTTGCCAAAACT IRDR-R Forward GCTGAAATGAATCATTCTCTCTACTATTATTCTGA Reverse AATTCCCTGTCTTAGGTCAGTTAGGA Probe TCACCACTTTATTTTAAGAATGTG Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 7 of 8 http://www.biomedcentral.com/content/supplementary/1759-8753-2-5-S1.TIFF Germany. 3Department of Human Genetics, University of Debrecen, Debrecen, Hungary. Authors’ contributions OK established the HEK clone; OK and VK optimized the real-time PCR and performed copy-number measurements; AS, ZE and ÁA established the HUES9 clones; GV helped in FACS measurements; LM measured copy numbers in HeLa clones; ZsI and ZI gave technical help and advices with the SB transposon work; BS provided financial support and discussed the data; and TIO designed the overall strategy, analyzed the data and wrote the paper. Competing interests The authors declare that they have no competing interests. Received: 5 November 2010 Accepted: 3 March 2011 Published: 3 March 2011 References 1. Ivics Z, Izsvak Z: Transposons for gene therapy! Curr Gene Ther 2006, 6:593-607. 2. VandenDriessche T, Ivics Z, Izsvak Z, Chuah MK: Emerging potential of transposons for gene therapy and generation of induced pluripotent stem cells. Blood 2009, 114:1461-1468. 3. Claeys Bouuaert C, Chalmers RM: Gene therapy vectors: the prospects and potentials of the cut-and-paste transposons. Genetica 2010, 138:473-484. 4. Izsvak Z, Ivics Z: Sleeping beauty transposition: biology and applications for molecular therapy. Mol Ther 2004, 9:147-156. 5. Grabundzija I, Irgang M, Mates L, Belay E, Matrai J, Gogol-Doring A, Kawakami K, Chen W, Ruiz P, Chuah MK, VandenDriessche T, Izsvák Z, Ivics Z: Comparative analysis of transposable element vector systems in human cells. Mol Ther 2010, 18:1200-1209. 6. Hackett PB, Largaespada DA, Cooper LJ: A transposon and transposase system for human application. Mol Ther 2010, 18:674-683. 7. Williams DA: Sleeping beauty vector system moves toward human trials in the United States. Mol Ther 2008, 16:1515-1516. 8. Mates L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z: Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 2009, 41:753-761. 9. Ryder E, Russell S: Transposable elements as tools for genomics and genetics in Drosophila. Brief Funct Genomic Proteomic 2003, 2:57-71. 10. Mates L, Izsvak Z, Ivics Z: Technology transfer from worms and flies to vertebrates: transposition-based genome manipulations and their future perspectives. Genome Biol 2007, 8(Suppl 1):S1. 11. Bian Q, Belmont AS: BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression. Nucleic Acids Res 2010, 38:e127. 12. Sivalingam J, Krishnan S, Ng WH, Lee SS, Phan TT, Kon OL: Biosafety assessment of site-directed transgene integration in human umbilical cord-lining cells. Mol Ther 2010, 18:1346-1356. 13. Huang X, Haley K, Wong M, Guo H, Lu C, Wilber A, Zhou X: Unexpectedly high copy number of random integration but low frequency of persistent expression of the Sleeping Beauty transposase after trans delivery in primary human T cells. Hum Gene Ther 2010, 21:1577-1590. 14. Wicks SR, de Vries CJ, van Luenen HG, Plasterk RH: CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans. Dev Biol 2000, 221:295-307. 15. Devon RS, Porteous DJ, Brookes AJ: Splinkerettes–improved vectorettes for greater efficiency in PCR walking. Nucleic Acids Res 1995, 23:1644-1645. 16. Moeller F, Nielsen FC, Nielsen LB: New tools for quantifying and visualizing adoptively transferred cells in recipient mice. J Immunol Methods 2003, 282:73-82. 17. Wang LJ, Chen YM, George D, Smets F, Sokal EM, Bremer EG, Soriano HE: Engraftment assessment in human and mouse liver tissue after sex- mismatched liver cell transplantation by real-time quantitative PCR for Y chromosome sequences. Liver Transpl 2002, 8:822-828. 18. Ballester M, Castello A, Ibanez E, Sanchez A, Folch JM: Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals. Biotechniques 2004, 37:610-613. 19. Joshi M, Keith Pittman H, Haisch C, Verbanac K: Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice. Biotechniques 2008, 45:247-258. 20. Ivics Z, Hackett PB, Plasterk RH, Izsvak Z: Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 1997, 91:501-510. 21. Baer M, Nilsen TW, Costigan C, Altman S: Structure and transcription of a human gene for H1 RNA, the RNA component of human RNase P. Nucleic Acids Res 1990, 18:97-103. 22. Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB: Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol 2002, 318:1221-1235. 23. Chung S, Andersson T, Sonntag KC, Bjorklund L, Isacson O, Kim KS: Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 2002, 20:139-145. 24. Liew CG, Draper JS, Walsh J, Moore H, Andrews PW: Transient and stable transgene expression in human embryonic stem cells. Stem Cells 2007, 25:1521-1528. 25. Xia X, Zhang Y, Zieth CR, Zhang SC: Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter- dependent manner. Stem Cells Dev 2007, 16:167-176. 26. Orban TI, Seres L, Ozvegy-Laczka C, Elkind NB, Sarkadi B, Homolya L: Combined localization and real-time functional studies using a GFP- tagged ABCG2 multidrug transporter. Biochem Biophys Res Commun 2008, 367:667-673. 27. Orban TI, Apati A, Nemeth A, Varga N, Krizsik V, Schamberger A, Szebenyi K, Erdei Z, Varady G, Karaszi E, Homolya L, Német K, Gócza E, Miskey C, Mátés L, Ivics Z, Izsvák Z, Sarkadi B: Applying a “double-feature” promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery. Stem Cells 2009, 27:1077-1087. 28. Izsvak Z, Khare D, Behlke J, Heinemann U, Plasterk RH, Ivics Z: Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition. J Biol Chem 2002, 277:34581-34588. 29. Apati A, Orban TI, Varga N, Nemeth A, Schamberger A, Krizsik V, Erdelyi- Belle B, Homolya L, Varady G, Padanyi R, Karászi E, Kemna EW, Német K, Sarkadi B: High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells. Biochim Biophys Acta 2008, 1778:2700-2709. doi:10.1186/1759-8753-2-5 Cite this article as: Kolacsek et al.: Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers. Mobile DNA 2011 2:5. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Kolacsek et al. Mobile DNA 2011, 2:5 http://www.mobilednajournal.com/content/2/1/5 Page 8 of 8 http://www.ncbi.nlm.nih.gov/pubmed/17073604?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19471016?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19471016?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19471016?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19649713?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19649713?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14759798?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14759798?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20372108?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20372108?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20104209?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20104209?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18725873?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18725873?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19412179?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19412179?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/15239944?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/15239944?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18047686?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18047686?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18047686?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20385594?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20385594?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20424600?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20424600?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20424600?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20528476?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20528476?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20528476?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/20528476?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10790327?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10790327?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/10790327?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/7784225?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/7784225?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14604542?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/14604542?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12200785?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12200785?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12200785?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/15517974?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/15517974?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/15517974?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18778249?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18778249?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18778249?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/9390559?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/9390559?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/9390559?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/2308839?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/2308839?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12083513?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12083513?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12083513?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/11897870?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/11897870?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/11897870?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/17379764?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/17379764?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/17348812?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/17348812?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/17348812?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18182157?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18182157?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19415778?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19415778?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/19415778?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12082109?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12082109?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/12082109?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18793608?dopt=Abstract http://www.ncbi.nlm.nih.gov/pubmed/18793608?dopt=Abstract Abstract Background Results Conclusions Background Results and Discussion Conclusions Methods Cell-culture maintenance and creation of clones Transfection and transposition Flow cytometry Genomic DNA isolation, transposon display and Southern/dot blotting Quantitative real-time PCR Acknowledgements Author details Authors' contributions Competing interests References << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Gray Gamma 2.2) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /Warning /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 15 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 15 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.76 /HSamples [2 1 1 2] /VSamples [2 1 1 2] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 15 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 15 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /CHS /CHT /DAN /DEU /ESP /FRA /ITA (Utilizzare queste impostazioni per creare documenti Adobe PDF adatti per visualizzare e stampare documenti aziendali in modo affidabile. I documenti PDF creati possono essere aperti con Acrobat e Adobe Reader 5.0 e versioni successive.) /JPN /KOR /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken waarmee zakelijke documenten betrouwbaar kunnen worden weergegeven en afgedrukt. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /PTB /SUO /SVE /ENU (Use these settings to create Adobe PDF documents suitable for reliable viewing and printing of business documents. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice