The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells Nynne Sharma, Anne Kruse Hollensen, Rasmus O. Bak, Nicklas Heine Staunstrup, Lisbeth Dahl Schrøder, Jacob Giehm Mikkelsen* Department of Biomedicine, Aarhus University, Aarhus, Denmark Abstract DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 59-HS4 chicken b-globin (cHS4) insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells. Citation: Sharma N, Hollensen AK, Bak RO, Staunstrup NH, Schrøder LD, et al. (2012) The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells. PLoS ONE 7(10): e48421. doi:10.1371/journal.pone.0048421 Editor: Ferenc Mueller, University of Birmingham, United Kingdom Received June 7, 2012; Accepted September 25, 2012; Published October 26, 2012 Copyright: 2012 Sharma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The Danish Council for Independent Research, Medical Sciences (http://en.fi.dk/councils-commissions/the-danish-council- for-independent-research/scientific-research-councils/medical-sciences); The Lundbeck Foundation, (http://www.lundbeckfoundation.com); The Novo Nordisk Foundation (http://www.novonordiskfonden.dk/en/); The Danish National Advanced Technology Foundation (http://hoejteknologifonden.dk/en); The Foundation of 17-12-1981 (http://hoejteknologifonden.dk); Kgl. Hofbuntmager Aage Bangs Foundation (http://www.danderm-pdv.is.kkh.dk/h11u-5.htm); Helga and Peter Kornings Foundation (http://www.korningfonden.dk); Novo Scholarship Programme in Biotechnology and Pharmaceutical Sciences (http://www.novozymes. com); and the EU (EU-FP6-STREP, contract number 018961). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors received funding from The Lundbeck Foundation and the Novo Scholarship Programme in Biotechnology and Pharmaceutical Sciences. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: giehm@hum-gen.au.dk Introduction DNA transposons are mobile DNA elements with a natural ability to integrate genetic material into genomic DNA. Consisting of only two parts, a transposon element defined by inverted terminal repeat sequences and a transposase enzyme mediating excision and reintegration of the transposon element, DNA transposons can easily be transformed into plasmid-based gene vector systems. Transposons have long been used for gene transfer applications in invertebrate model organisms, such as Drosophila and Caenorhabditis elegans [1,2], but elements with efficient transposition in mammalian cells have been in high demand for biomedical and therapeutic applications. Reconstruction of Sleeping Beauty (SB), a Tc1/mariner element assembled from inactive salmonid fish transposon sequences, revealed the first DNA transposon vector reported to have high activity in vertebrate cells [3]. Since its resurrection, the SB system has proven to be active in a wide range of vertebrate species, which has made it a widely used non-viral tool for transgenesis and insertional mutagenesis studies [4,5]. In addition, observations of long-term gene expression after SB-mediated delivery in human primary cell types (including CD34+ [6,7,8], primary T [9,10,11,12], and embryonic stem cells [13,14]), have made the SB transposon a highly studied vector system for gene therapy applications [15,16]. In consequence, the first clinical trial utilizing SB-directed gene insertion has recently been initiated for adoptive immunotherapy treatment of patients with B-cell malignancies [17]. Since the re-activation of the SB transposon other transposable elements, capable of high-efficient transposition in mammalian cells, have been discovered. Amongst these are two naturally active elements, the piggyBac (PB) transposon, originally isolated from the cabbage looper moth Trichoplusia ni [18,19], and the Tol2 transposon, isolated from the genome of the Japanese medaka fish Oryzias latipes [20]. PB, the founding member of the piggyBac PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48421