MAR Elements and Transposons for Improved Transgene Integration and Expression

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.

A chimeric HS4 insulator–scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells

“…Matrix attachment regions can increase expression levels of the transgene in stably transfected CHO cells . However, the characteristics and mechanism of MARs function have not been elucidated, and further studies are needed to develop improved methods for transgene expression.…”

“…Scaffold/matrix attachment regions (S/MARs) can block transgene silencing and increase transgene expression levels and stability in host cells . In addition, S/MARs can also reduce variations in transgene expression among different cells to some extent, and the rate of transgene genomic integration can be increased . However, some reports have shown that S/MARs have inconsistent effects on transgene expression .…”

Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

“…In experiments where Foxa1 was overexpressed, the CMV/EF1alpha promoter was replaced by a minimal cytomegalovirus (CMV) promoter for both Foxa1 and GFP expressions. The piggyBac transposase expression vector (pCS2+U5V5PBU3) was previously described (Ley et al, 2013).…”

“…The piggyBac transposase expression vector (pCS2+U5V5PBU3) was previously described (Ley et al, 2013).…”

Recombinant protein production from stable mammalian cell lines and pools

“…Furthermore, both SB and PB have been used to construct cell lines for inducible transgene expression under the control of the Tet-On system [37,38 ,40]. Although PB-derived cell lines often have fairly constant levels of protein production over time in the absence of selection, the addition of a MAR element to the donor vector reduced the effects of gene silencing [41,42]. Besides the transposon systems mentioned here, others have recently been introduced under the trade name Leap-In TM (DNA2.0, Menlo Park, CA).…”