MAR Elements Research Articles

Current Landscape and Emerging Opportunities of Gene Therapy with Non-viral Episomal Vectors.

Gene therapy has proven to be extremely beneficial in the management of a wide range of genetic disorders for which there are currently no or few effective treatments. Gene transfer vectors are very significant in the field of gene therapy. It is possible to attach a non-viral attachment vector to the donor cell chromosome instead of integrating it, eliminating the negative consequences of both viral and integrated vectors. It is a safe and optimal express vector for gene therapy because it does not cause any adverse effects. However, the modest cloning rate, low expression, and low clone number make it unsuitable for use in gene therapy. Since the first generation of non-viral attachment episomal vectors was constructed, various steps have been taken to regulate their expression and stability, such as truncating the MAR element, lowering the amount of CpG motifs, choosing appropriate promoters and utilizing regulatory elements. This increases the transfection effectiveness of the non-viral attachment vector while also causing it to express at a high level and maintain a high level of stability. A vector is a genetic construct commonly employed in gene therapy to treat various systemic disorders. This article examines the progress made in the development of various optimization tactics for nonviral attachment vectors and the future applications of these vectors in gene therapy.

Precipitation‐Driven Pedogenic Weathering of Volcaniclastics on Early Mars

AbstractCompositional stratigraphy, generally composed of Al‐rich clay minerals overlying Fe/Mg‐rich clay minerals, is observed in many locations on Mars. Here we describe the occurrence of such mineralogical stratigraphy in settings where the protoliths are almost certainly pyroclastic materials. One such example includes altered rocks high on the summit and flanks of explosive volcanoes in Thaumasia Planum. These clay‐bearing deposits are most consistent with precipitation‐driven weathering of ash deposits. Considering explosive volcanism was pervasive in the Noachian, the early sedimentary record of Mars in some locations is likely dominated by glassy, fragmented, porous, chemically reactive materials with highly specific surface area. These pyroclastic deposits were potentially a critical geological component linking clay minerals to elements of Mars’ climate, weathering, and sedimentary puzzle.

Distribution of Interplanetary Dust Detected by the Juno Spacecraft and Its Contribution to the Zodiacal Light

AbstractThe Solar System is home to a cloud of dust that orbits the Sun and makes its presence known by virtue of scattered light (Zodiacal Light) that can be seen after dusk and before dawn. Within this cloud are bands of dust orbiting near the ecliptic plane, evidenced by an excess of scattered light at discrete ecliptic latitudes. Dedicated dust detectors borne by spacecraft in transit of the solar system have detected few such particles of the appropriate size owing to limited detector aperture and sparsity of the population. Thus, the distribution, origin, and orbital evolution of the dust in these bands remains a mystery. A star camera aboard the Juno spacecraft traveling from Earth to Jupiter recorded interplanetary dust impacts on the spacecraft in numbers sufficient to characterize the spatial distribution of such particles for the first time. The observed distribution is consistent with a primary source of dust particles sharing the Mars orbit plane between Earth and the 4:1 resonance with Jupiter. We propose that the primary distribution is scattered by orbital resonances with Jupiter via the Kozai‐Lidov (KL) effect into a secondary population at higher inclination to the ecliptic. The measured dust distribution, occupying a volume uniquely determined by the orbital elements of Mars and KL scattering, accounts for the observed variation of the Zodiacal Light with ecliptic latitude. Our results provide a compelling alternative to the prevailing theory of the origin and evolution of interplanetary dust observed at low ecliptic latitudes.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the β-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080cells.

The rotational elements of Mars and its satellites

The International Astronomical Union (IAU) defines planet and satellite coordinate systems relative to their axis of rotation and the angle about that axis. The rotational elements of the bodies are the right ascension and declination of the rotation axis in the International Celestial Reference Frame and the rotation angle, W, measured easterly along the body's equator. The IAU specifies the location of the body's prime meridian by providing a value for W at epoch J2000. We provide new trigonometric series representations of the rotational elements of Mars and its satellites, Phobos and Deimos. The series for Mars are from a least squares fit to the rotation model used to orient the Martian gravity field. The series for the satellites are from a least squares fit to rotation models developed in accordance with IAU conventions from recent ephemerides.

Divergent Levels of Marker Chromosomes in an hiPSC-Based Model ofPsychosis.

SummaryIn the process of generating presumably clonal human induced pluripotent stem cells (hiPSCs) from two carriers of a complex structural rearrangement, each having a psychotic disorder, we also serendipitously generated isogenic non-carrier control hiPSCs, finding that the rearrangement occurs as an extrachromosomal marker (mar) element. All confirmed carrier hiPSCs and differentiated neural progenitor cell lines were found to be mosaic. We caution that mar elements may be difficult to functionally evaluate in hiPSC cultures using currently available methods, as it is difficult to distinguish cells with and without mar elements in live mosaic cultures.

Nuclear matrix protein SMAR1 control regulatory T-cell fate during inflammatory bowel disease (IBD).

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Transcription factor Foxp3, a positive regulator of Treg cell differentiation, has been studied to some extent. Signal transducer and activator of transcription factor 3 (STAT3) is known to negatively regulate Foxp3. It is not clear how STAT3 is regulated during Treg differentiation. We show that SMAR1, a known transcription factor and tumor suppressor, is directly involved in maintaining Treg cell fate decision. T-cell-specific conditional knockdown of SMAR1 exhibits increased susceptibility towards inflammatory disorders, such as colitis. The suppressive function of Treg cells is compromised in the absence of SMAR1 leading to increased T helper type 17 (Th17) differentiation and inflammation. Compared with wild-type, the SMAR1−/− Treg cells showed increased susceptibility of inflammatory bowel disease in Rag1−/− mice, indicating the role of SMAR1 in compromising Treg cell differentiation resulting in severe colitis. We show that SMAR1 negatively regulate STAT3 expression favoring Foxp3 expression and Treg cell differentiation. SMAR1 binds to the MAR element of STAT3 promoter, present adjacent to interleukin-6 response elements. Thus Foxp3, a major driver of Treg cell differentiation, is regulated by SMAR1 via STAT3 and a fine-tune balance between Treg and Th17 phenotype is maintained.

Impact of using different promoters and matrix attachment regions on recombinant protein expression level and stability in stably transfected CHO cells.

High expression level and long-term expression stability are required for therapeutic protein production in mammalian cells. Three commonly used promoters from the simian virus 40 (SV40), the CHO elongation factor 1α gene (EF1α), and the human cytomegalovirus major immediate early gene (CMV) and two matrix attachment regions from the chicken lysozyme gene (cMAR) and the human interferon β (iMAR) were evaluated for enhancing recombinant gene expression level and stability in stably transfected CHO cells. In the absence of MAR elements, the SV40 promoter gave lower expression level but higher stability than the EF1α promoter and the CMV promoter. The inclusion of MAR elements did not increase the integrated gene copies for all promoters but did enhance expression level for only the SV40 promoter. The enhanced gene expression was due to an increase in mRNA levels. Neither MAR elements enhance gene expression stability during long-term culture. The combinations of SV40 promoter and MAR elements are the best for obtaining both high expression level and stability. The information presented here would be valuable to those developing vectors for generation of CHO cell lines with stable and high productivity.

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

BackgroundThe in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.ResultsIn this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression.ConclusionsThis study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

Near distance approximation in astrodynamical applications of Lambert’s theorem

The smallness parameter of the approximation method is defined in terms of the non-dimensional initial distance between target and chaser satellite. In the case of a circular target orbit, compact analytical expressions are obtained for the interception travel time up to third order. For eccentric target orbits, an explicit result is worked out to first order, and the tools are prepared for numerical evaluation of higher order contributions. The possible transfer orbits are examined within Lambert’s theorem. For an eventual rendezvous it is assumed that the directions of the angular momenta of the two orbits enclose an acute angle. This assumption, together with the property that the travel time should vanish with vanishing initial distance, leads to a condition on the admissible initial positions of the chaser satellite. The condition is worked out explicitly in the general case of an eccentric target orbit and a non-coplanar transfer orbit. The condition is local. However, since during a rendezvous maneuver, the chaser eventually passes through the local space, the condition propagates to non-local initial distances. As to quantitative accuracy, the third order approximation reproduces the elements of Mars, in the historical problem treated by Gauss, to seven decimals accuracy, and in the case of the International Space Station, the method predicts an encounter error of about 12 m for an initial distance of 70 km.

Mar, a MITE family of hAT transposons in Drosophila

BackgroundMiniature inverted-repeat transposable elements (MITEs) are short, nonautonomous DNA elements flanked by subterminal or terminal inverted repeats (TIRs) with no coding capacity. MITEs were originally recognized as important components of plant genomes, where they can attain extremely high copy numbers, and are also found in several animal genomes, including mosquitoes, fish and humans. So far, few MITEs have been described in Drosophila.ResultsHerein we describe the distribution and evolution of Mar, a MITE family of hAT transposons, in Drosophilidae species. In silico searches and PCR screening showed that Mar distribution is restricted to the willistoni subgroup of the Drosophila species, and a phylogenetic analysis of Mar indicates that this element may have originated prior to the diversification of these species. Most of the Mar copies in D. willistoni present conserved target site duplications and TIRs, indicating recent mobilization of these sequences. We also identified relic copies of potentially full-length Mar transposon in D. tropicalis and D. willistoni. The phylogenetic relationship among transposases from the putative full-length Mar and other hAT superfamily elements revealed that Mar is placed into the recently determined Buster group of hAT transposons.ConclusionOn the basis of the obtained data, we can suggest that the origin of these Mar MITEs occurred before the subgroup willistoni speciation, which started about 5.7 Mya. The Mar relic transposase existence indicates that these MITEs originated by internal deletions and suggests that the full-length transposon was recently functional in D. willistoni, promoting Mar MITEs mobilization.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that >98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Using a combination of SAR element and enhancer to increase the expression level of hybrid genes in transgenic animals

A regulatory element consisting of a MAR element sequence of Drosophila heat shock genes and enhancer from a long terminal repeat of the mouse mammary tumor virus is created for studying the possibility of increasing the expression level of hybrid genes in transgenic animals. Two lines of transgenic mice containing the described regulatory element and hybrid gene intended for expressing human beta interferon in milk of transgenic animals are obtained by the method of coinjection into the pronuclei of murine zygotes. It is shown that the use of the given regulatory element doesn’t lead to an increase of activity of human beta interferon in milk compared with transgenic rabbits containing only the hybrid gene for expressing beta interferon. Furthermore, a decrease of tissue-specific expression of beta interferon is observed with the use of the regulatory element.

486. Sleeping Beauty Expression in BOEC Is Significantly Increased with the β-Globin MAR Element

486. Sleeping Beauty Expression in BOEC Is Significantly Increased with the β-Globin MAR Element

Various Expression-Augmenting DNA Elements Benefit from STAR-Select, a Novel High Stringency Selection System for Protein Expression

The creation of highly productive mammalian cell lines often requires the screening of large numbers of clones, and even then expression levels are often low. Previously, we identified DNA elements, anti-repressor or STAR elements, that increase protein expression levels. These positive effects of STAR elements are most apparent when stable clones are established under high selection stringency. We therefore developed a very high selection system, STAR-Select, that allows the formation of few but highly productive clones. Here we compare the influence of STAR and other expression-augmenting DNA elements on protein expression levels in CHO-K1 cells. The comparison is done in the context of the often-used cotransfection selection procedure and in the context of the STAR-Select system. We show that STAR elements, as well as MAR elements induce the highest protein expression levels with both selection systems. Furthermore, in trans cotransfection of multiple copies of STAR and MAR elements also results in higher protein expression levels. However, highest expression levels are achieved with the STAR-Select selection system, when STAR elements or MARs are incorporated in a single construct. Our results also show that the novel STAR-Select selection system, which was developed in the context of STAR elements, is also very beneficial for the use of MAR elements.

Inclusion of a matrix-attached region in a 7SK pol III vector increases the efficiency of shRNA-mediated gene silencing in embryonic carcinoma cells

RNA interference is a widely used tool for analysis of gene function in mammalian cells. Stable knockdown of specific target genes can be maintained in cell lines and live organisms using vector-based delivery of short hairpins (shRNAs) driven by RNA polymerase III promoters. Here we describe a vector incorporating the human 7SK promoter for shRNA-mediated gene silencing in the P19 embryonic carcinoma stem cell line. Our preliminary experiments with the 7SK shRNA expression vector indicated that its activity could be hindered by random genomic integration. In order to counter this inhibitory mechanism, we inserted a matrix-attached region sequence to generate an episomal vector system. We compared the effects of insertion versus exclusion of the MAR sequence on the shRNA-mediated gene-specific silencing of the β- tubulin III and Cyclophilin A genes. While the MAR sequence is not strongly correlated with the episomal status of the expression vector, our studies indicate that inclusion of the MAR element significantly enhances the stability of shRNA-mediated gene silencing in the P19 stem cells.

Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix

The spatial organization of an ∼170 kb region of human chromosome 19, including CD22 and GPR40–GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix.

Mutant p53 proteins bind DNA in a DNA structure-selective mode

Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53.