Abstract
Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1–68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1–68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2–4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.
Highlights
Efficient gene transfer and expression for functional studies, protein production or gene and cell therapies usually requires reliable DNA delivery and transcription into target cells
To test whether adding a Matrix Attachment Region (MAR) element to the PB transposon may affect transposition efficiency and transgene expression, and to assess whether the location of the MAR in the construct had any influence on these effects, we designed a series of transposon donor constructs containing the green fluorescent protein (GFP) and puromycin resistance (Puro) gene, in which the MAR 1–68 or a control neutral spacer DNA sequence were inserted at different positions in the plasmid (Figure 1)
Most studies involving transposable vectors have relied on antibiotic resistance-based assays, which has not allowed a clear and quantitative disentanglement of transgene copy numberlinked effects from those elicited by variations of transgene expression levels
Summary
Efficient gene transfer and expression for functional studies, protein production or gene and cell therapies usually requires reliable DNA delivery and transcription into target cells. Non-viral vectors are associated with a reduced risk of insertional mutagenesis when compared to e.g. retroviral vectors for gene or cell therapies, and they are easier to produce [1] They typically require physical (e.g. electroporation) or chemical (e.g. cationic lipids) DNA transfer methods that are not applied in vivo, and they are less efficient than viral vectors when genomic integration of the transgene is necessary. Stable transfection relies on the selection of rare cells having integrated plasmid DNA into one or few genomic loci, as a result of the action of cellular DNA repair and recombination enzymes [2] This leads to the integration of multi-copy plasmid concatemers, usually as head-to-tail arrays [3,4]. It is expected to increase the probability of observing point mutations in one or few copies, which are often difficult to detect during early cell line characterization stages, and it has been associated to repeatinduced silencing events [5,6,7]
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