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The piggyBac inverted terminal repeats (ITR), the transposase coding sequence (PB), the bacterial origin of replication (Ori) and the ampicillin selection gene (Amp) schematically depict the plasmids used in this study. The puromycin resistance (Puro) and reporter green fluorescent protein (GFP) genes are illustrated by yellow and green arrows and the spacer or MAR 1–68 sequences are shown by white or blue boxes on the transposon donor plasmid derivatives.
Source publication
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 m...
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Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product. Additionally, the effects of transgene design across diverse cell types are not...
Nowadays, in most gene therapy trials virus based vectors are used because of their high efficiency. Nevertheless, safety risks like transformation of the cell by viral proteins, insertional mutagenesis, or innate immune reactions cannot be excluded. Basing on the idea of an ideal vector for gene therapy that is highly efficient but lacks these saf...
Citations
... Therefore, MARs also appear to be associated with gene expression levels. Additional MAR regions in promoters have been shown to enhance the expression of transgenes, also supporting a role for the nuclear matrix in gene expression 50,51 . Apart from MAR regions, the MAR-binding protein AHL29 acts as both a positive and a negative regulator of transcription to regulate petiole growth 37 , suggesting that AHLs also act as transcriptional activators. ...
The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.
... Insertion of MAR 1-68 into the center of the PB transposon does not affect transposition efficiency, whereas insertion into the edge of the transposon near the ITR side might interfere with transposase function and affect transposition efficiency. This is possibly due to the function alteration of transposase in targeting genomic locus and integrating the transposon sequence caused by MAR 1-68 (Ley et al., 2013). The addition of the 59-HS4 chicken b-globin (cHS4) insulator sequence in retina pigment epithelium cells leads to increased transgene expression levels for SB and PB vectors, respectively, but the cHS4 insulator did not show a long- term protective effect against the transgene silencing in retinal pigment epithelium cells (Sharma et al., 2012). ...
In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.
... 11,[13][14][15][16][17][18] Out of them, the MARs have been extensively studied and are often integrated as cis-acting elements into expression vectors. 19 MAR sequences are DNA regions that can link the nuclear matrix and are characterized by an AT-rich core surrounded by two flanking regions with binding sites for various factors (insulators, transcription factors, nuclear matrix protein, etc.). 20 For these reasons, MAR elements delimit topological domains, called chromatin loops, in which genes are protected from silencing and actively expressed. ...
... 11,21,46,57 These positive effects of the MAR element, reasonably due to its role of epigenetic element acting as boundary element and its ability to mediate transgene integration into permissive and active sites of the genome, prevents the elaborate work of identifying genomic hot spots prior to integration. 19,22,54 . However, it will be interesting to have data on LP integration sites by analyzing many more clones to determine if there are some preferential genomic insertion sites. ...
... The other landing pad vector harbors the same cMAR sequence followed by pCMV promoter, the AttB site for BxB1 recombinase containing a central dinucleotide mutation (GA instead of GT), DsRed reporter gene and a bhg polyA tail. The use of the MAR in this construct should help the integration of multiple copies of the landing pad into the host genome via concatemer formation, protect it from silencing due to position effects and improve the stability of the system.19,46 The orthogonal AttB sites forBxB1 can recombine only with the AttP sites carrying the matching central dinucleotide, allowing integration of GOI specifically at one or the other landing pad. ...
In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.
... The reference gene used in this study is the beta-2microglubulin (Data S1). The number of integrated transgene (Puro R or CMV promoter) was calculated relative to that of the reference beta-2microglobulin (B2M) gene present in 2 copies in the CHO genome (Ley et al., 2013), as previously described (Pfaffl, 2001;Karlen et al., 2007). Results represent data obtained from qPCR on the gDNA obtained from three independent transfections. ...
Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.
... Most importantly, the vector design can certainly be further improved. Some advancement was already achieved in enhancing expression of transgenes by insertion of matrix attachment regions (MARs; Ley et al. 2013). To minimize gene silencing, the utilization of flanking cHS4 DNA insulators was instrumental (Sharma et al. 2012). ...
Stable recombinant mammalian cells are of growing importance in pharmaceutical biotechnology production scenarios for biologics such as monoclonal antibodies, growth and blood factors, cytokines and subunit vaccines. However, the establishment of recombinant producer cells using classical stable transfection of plasmid DNA is hampered by low stable gene transfer efficiencies. Consequently, subsequent selection of transgenic cells and the screening of clonal cell populations are time- and thus cost-intensive. To overcome these limitations, expression cassettes were embedded into transposon-derived donor vectors. Upon the co-transfection with transposase-encoding constructs, elevated vector copy numbers stably integrated into the genomes of the host cells are readily achieved facilitating under stringent selection pressure the establishment of cell pools characterized by sustained and high-yield recombinant protein production. Here, we discuss some aspects of transposon vector technologies, which render these vectors promising candidates for their further utilization in the production of biologics.
... Matrix attachment regions (MARs) are cis-acting DNA elements and known to act as epigenetic regulatory sequences that increase gene expression [95]. Ley et al. [96] demonstrated that incorporation of human MAR 1-68 in a PB transposon cassette was beneficial for reduced silencing effects and caused increased transgene expression in cultured cells. This finding was also confirmed by Zhao et al. (2017) [97]. ...
In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. piggyBac (PB), one of three transposons (PB, Sleeping Beauty (SB), and Tol2) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.
... To avoid mutual interaction or transgene silencing, genomic insulators were often chosen to be inserted among transcriptional units or flanked at both ends of expressing cassettes. So far, several insulators or elements, such as the chicken hypersensitive site 4 (cHS4) insulator or scaffold or matrix attachment regions elements, have been found to play positive roles in enhancing transgene expression or blocking transgene silencing in previous studies [14][15][16]. The combination of HS4 and scaffold attachment region elements (chimeric insulator) was also reported to enhance transgene expression across different cell lines, such as human pluripotent stem cells, Chinese hamster ovary cells and 293T cells [17][18][19]. ...
Synthetic biology circuits are often constructed with multiple gene expression units assembled in close proximity, and can be used to perform complex functions in embryonic stem cells (ESCs). However, mutual interference between transcriptional units has not been well studied in mouse ESCs. To assess the efficiency of insulators at suppressing promoter interference in mouse ESCs, we utilized an evaluation scheme, in which a tunable TRE promoter is connected to a constant Nanog promoter. Chicken hypersensitive site 4 (cHS4) insulator, widely used both for enhancer blocking and barrier insulation in vitro and in vivo, was positioned between the two expression units for assessment. By inserting the cassette into various loci of the mouse ESC genome with PiggyBac transposon，we were able to quantitatively examine the protective effect of cHS4 by gradually increasing the transcriptional activity of the TRE promoter with doxycycline, and then measuring the transcriptional activity of the Nanog promoter. Our results indicate that the cHS4 insulator has minimal insulating effects on promoter interference in mouse ESCs. Further studies show that the cHS4 insulation effect may be promoter‐specific and related to interaction with CCCTC‐binding factor (CTCF) mediated loop formation. In addition, we also compared DNA transposition and transgene expression with or without the cHS4 insulator using well‐established ESC reporters. The results indicate that cHS4 has no apparent effects on DNA transposition and transgene expression levels, but exerts modest protective effects on long‐term transgene silencing.
... Here we report that the expression of specific combinations of these engineering target genes yields increased cell density, viability, and specific productivity in fed-batch cultures, resulting in higher production of easy-to-express as well as difficult-to-express therapeutic proteins and decreased reactive oxygen species (ROS), providing novel avenues towards highly efficient therapeutic protein production. The piggyBac transposase expression vector (pCS2+U5V5PBU3) was previously described (Ley et al., 2013). ...
Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy‐to‐express antibody, whereas Erp27 and Erp57 co‐overexpression increased cell density, viability, and the yield of difficult‐to‐express proteins. Foxa1 overexpression increased cell density, cell viability, and easy‐ and difficult‐to‐express protein yields, whereas it decreased reactive oxygen species late in fed‐batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult‐ and/or easy‐to‐express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.
... Through the binding of MARs to the nuclear matrix, the chromatin can form an independent loop structure, regulate gene transcription and expression and reduce transgene silencing due to positional effects. Previous studies have reported that MARs improve transgene expression levels in mammalian cell expression systems and reduce the differences of expression levels amongst transgenic individuals, thereby inhibiting transgene silencing (Sjeklocha et al. 2011;Ley et al. 2013;Chang et al. 2014). ...
... In brief, MAR X-29 was subcloned into the upstream of the CMV promoter of pIRES-EGFP vector, and different lengths (500, 1000, 1500, 2000 and 2500 bp) of neutral spacer DNA sequences (NCBI RefSeq: NM_011682.4) were PCR-amplified from the mouse utrophin cDNA (Ley et al. 2013) and inserted between the MAR and the CMV promoter to obtain expression vectors containing MAR with different distances (Fig. 1). All cloning procedures were performed using standard molecular techniques (Sambrook and Russell 2001). ...
... Moreover, the sequence was from eGFP, which cannot effectively reflect the characteristics of the spacer DNA sequence. In the present study, a control neutral spacer DNA sequence, which cannot improve transgene expression, was inserted between the MAR and the CMV promoter of the construct to systematically explore the characteristics of different MAR-mediated distances in regulating transgene expression (Ley et al. 2013). Five different lengths (500, 1000, 1500, 2000 and 2500 bp) (Fig. S1) were used to explore the optimum length. ...
Objectives
Previously, we have found that the matrix attachment region (MAR) may confer a ‘distance effect’ on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism.
Results
Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers.
Conclusions
A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.
... Most importantly, the vector design can certainly be further improved. Some advancement was already achieved in enhancing expression of transgenes by insertion of matrix attachment regions (MARs; Ley et al. 2013). To minimize gene silencing, the utilization of flanking cHS4 DNA insulators was instrumental (Sharma et al. 2012). ...
Stable mammalian, namely human, suspension cell lines play a pivotal role in red biotechnology production scenarios for the generation of state-of-the-art biologics. However, selection of genetically modified and highly productive cell populations – prior to the establishment of clonal lines – is often challenging. To overcome this limitation, we first describe an optimized transient transfection protocol using the inexpensive reagent polyethylenimine (PEI) and human 293F cells. Transposon donor vectors derived from Sleeping Beauty encompassing a cassette with the reporter gene encoding for the green fluorescent protein (GFP) coupled with an internal ribosome entry site (IRES) to the expression of puromycin-resistance are employed to readily detect transfected cells. Upon stable transfection in the presence and absence of transposase expression, respectively, and subsequent antibiotic selection, GFP expression using flow cytometry analysis, cell viability, and cell density can be examined over a range of up to 3 weeks. Owing to the integration of high vector copy numbers into the target cell genome, transposase-mediated transposition of transposon donor vectors is instrumental in the faster establishment of recombinant cell population as compared to the classical stable transfection of plasmid DNA.
