&p.1:Abstract We evaluated the survival, transgene produc- tion, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing mu- rine sequence nontranscribed spacer (NTS) to the plasm- ids increased transfection efficiency, survival, and trans- gene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design. &kwd:Key words Plasmid vector · Lipofection · Transgene expression · Gene therapy · Nontranscribed Spacer Abbreviations CMV Cytomegalovirus · G-CSF Granulocyte-colony stimulating factor · IRES Internal ribosomal entry sequence · NTS Nontranscribed spacer&bdy: Introduction To improve gene therapy it is important to enhance the integration of foreign DNA into suitable cells and to en- sure a high level of transcription of the transfected trans- gene. While retroviral gene transfer with its compara- tively high transduction efficiency offers many advanta- ges, nonviral plasmid vector systems can be used effec- tively ex vivo when cultured cells are used that can be selected and cloned according to criteria considered opti- mal for in vivo therapy upon administration of transfec- ted cells [1–6]. To optimize gene transfer and transcrip- tion of recombinant genes in a plasmid-based gene trans- fer model we investigated the effects of selection pres- sure, copy number, and various vector backbones on transfection and transcription. Several events can result in high expression of the plasmid’s transcriptional units. First, more than one copy of plasmid DNA per cell might in some systems assure more transcriptional units leading to higher protein pro- duction per cell. Second, integration can occur at a very active region of the chromosome and a plasmid-borne promoter acitvity can be multiplied by cellular enhance- rs. Third, the plasmid itself activates via plasmid-encod- ed sequences the chromosome at the locus of integration and thus activates transcription per se. We designed a variety of plasmids addressing these questions. The plasmids contained a selectable neomycin resistance gene marker [7], a granulocyte colony stimu- lating factor (G-CSF) expression cassette [8], and a non- transcribed spacer (NTS) sequence. This NTS sequence derived from murine rDNA is apparently involved in nu- cleosome assembly and plasmid amplification. Plasmids containing this sequence have been shown both in mu- rine and in some human cells to raise integration at high copy numbers [9, 10]. We investigated the effects of the NTS sequence on transgene expression, studying both the quantity of ami- noglycoside phosphotransferase gene (neomycin resis- tance) expression at increasing neomycin concentrations and the expression of the gene of interest by quantitating the gene product by enzyme-linked immunosorbent as- say. G-CSF was chosen as the gene of interest in view of its ability to ameliorate chemotherapy-induced cytopenia in an in vivo model system [11]. Since we have previous- ly shown that an internal ribosomal entry sequence (IRES) [12, 13] allowing the translation of the G-CSF cDNA and the neomycin resistance gene from the same RNA, tightly linking the two genes, leads to enhanced transgene expression [14], we investigated the effect of P. Kulmburg ( ✉ ) · H. Veelken · F.M. Rosenthal A. Mackensen · A. Lindemann · R. Mertelsmann Department of Internal Medicine I (Hematology/Oncology), University of Freiburg Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Germany C. Schmoor Institut für Medizinische Biometrie und Medizinische Informatik, University of Freiburg, Stefan-Meier-Strasse 26, D-79104 Freiburg, Germany&/fn-block: J Mol Med (1997) 75:223–228 © Springer-Verlag 1997 ORIGINAL ARTICLE &roles:P. Kulmburg · H. Veelken · C. Schmoor F.M. Rosenthal · A. Mackensen · A. Lindemann R. Mertelsmann NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line &misc:Received: 1 October 1996 / Accepted: 27 November 1996