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Eye color phenotypes of Drosophila transformants expressing mini-white from white enhancerless vectors, series 2. Four-day-old females heterozygous for each of the different, single-copy P-element insertions were classified as having light yellow (phenotypic class I), yellow (II), light orange (III), orange (IV), or red (V) eyes. A representative of each of the phenotypes obtained with the different white vectors is shown, and the number of transformed lines with that phenotype is indicated below each picture. (a) ATRmwS transformants had yellow (II) or light orange (III) eyes. (b) apoB5MmwS transformants and (c) apoBmwS transformants were widely distributed in the light yellow (I), yellow (II), light orange (III), and red (V) phenotypic classes.
Source publication
Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have ins...
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... vector in which the ATR MAR was inserted upstream of the white transcrip- tion unit (ATRMmwS [Fig. 1g]) was prepared and used to transform w Drosophila embryos. Nine of 10 transformed lines contained single-copy ATRMmwS insertions (data not shown). Seven of the nine single-copy lines had yellow (II) eyes, and two had light orange (III) eyes (Fig. 5a). This distri- bution of eye color phenotypes was similar to that observed in apoB3MmwS transformants (Fig. 4c), suggesting that the hu- man ATR MAR has insulating properties similar to those of the apoB 3 ...
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... unit in the white enhancerless P- element vector (Fig. 1h). Twelve apoB5MmwS transformants were obtained; eight of these contained single transgene inser- tions and four contained double transgene insertions (Fig. 2b). The eye color phenotypes of the eight single-copy transfor- mants varied considerably, ranging from light yellow (I) to red (V) (Fig. 5b). This range of phenotypes, without enrichment for any particular phenotypic class, was much like that seen in the mwS transformants (Fig. 4a), which do not contain an insulator element upstream of white. These results indicate that the putative apoB 5 MAR does not function as an insu- lator element in Drosophila. This observation ...
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... was inserted up- stream of the white transcription unit (apoBmwS [Fig. 1i]). This human DNA fragment is clearly devoid of matrix-binding activity. Eight of 11 transgenic lines containing apoBmwS had single-copy transgene insertions. The eye color phenotypes of these eight transformed lines varied widely, ranging from pale yellow (I) to red (V) (Fig. 5c). These results were similar to those obtained with the other position-sensitive P elements, mwS (Fig. 3a) and apoB5MmwS (Fig. 5b). Therefore, this DNA fragment, which contains intron and exon sequences from the human apoB gene, does not function as an insulator element in Drosophila. Thus, human DNA does not have in- trinsic insulating ...
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... clearly devoid of matrix-binding activity. Eight of 11 transgenic lines containing apoBmwS had single-copy transgene insertions. The eye color phenotypes of these eight transformed lines varied widely, ranging from pale yellow (I) to red (V) (Fig. 5c). These results were similar to those obtained with the other position-sensitive P elements, mwS (Fig. 3a) and apoB5MmwS (Fig. 5b). Therefore, this DNA fragment, which contains intron and exon sequences from the human apoB gene, does not function as an insulator element in Drosophila. Thus, human DNA does not have in- trinsic insulating activity in this assay, nor is the insulating phenotype due to a distance effect between the white transcrip- tion units and ...
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... phenotypic classes. Thus, the eye color pheno- types of white transformants are very sensitive indicators of white gene expression, which makes this system particularly useful as a position effect assay. Among the white enhancerless transformants, the apoB 3 MAR and the ATR MAR both enriched for yellow (II) to light orange (III) transformants (Fig. 4c and 5a). These phenotypes differed only about 1.2-fold in eye pigment expression. These observations provide further support for the conclusion that these two human MARs can function as insulator elements in ...
Citations
... Several studies were also carried out to investigate MARs in vertebrates such as humans, mice, chickens and Chinese hamsters [16][17][18][19]. The influence of MARs in gene expression and their successful integration and regulation of transgenic genes were also determined [19][20][21][22]. ...
... The role of transcription factors (TFs) and their binding to DNA sequences such as the MAR regions and the regulatory elements: promoters, enhancers, ORIs, and silencers, to aid the regulation of gene expression in eukaryotes has also been explored by previous studies [63]. The use of MARs in transfected cells, regardless of species, demonstrated their role in epigenetic maintenance and consistent insulating activity that allows for relatively stable gene expression [20,21]. MARs were found to enhance the transgene expression and the MAR assisted transcriptional activation of these transgenes involved AT-rich sequences and specific TF binding motifs [22]. ...
Background
Chromatin architecture is critical for gene expression during development. Matrix attachment regions (MARs) control and regulate chromatin dynamics. The position of MARs in the genome determines the expression of genes in the organism. In this study, we set out to elucidate how MARs temporally regulate the expression of the fibroin heavy chain (FIBH) gene during development. We addressed this by identifying MARs and studying their distribution and differentiation, in the posterior silk glands of Bombyx mori during 5th instar development.
Results
Of the MARs identified on three different days, 7.15% MARs were common to all 3 days, whereas, 1.41, 19.27 and 52.47% MARs were unique to day 1, day 5, and day 7, respectively highlighting the dynamic nature of the matrix associated DNA. The average chromatin loop length based on the chromosome wise distribution of MARs and the distances between these MAR regions decreased from day 1 (253.91 kb) to day 5 (73.54 kb) to day 7 (39.19 kb). Further significant changes in the MARs in the vicinity of the FIBH gene were found during different days of 5 th instar development which implied their role in the regulation and expression of the FIBH gene.
Conclusions
The presence of MARs in the flanking regions of genes found to exhibit differential expression during 5 th instar development indicates their possible role in the regulation of their expression. This reiterates the importance of MARs in the genomic functioning as regulators of the molecular mechanisms in the nucleus. This is the first study that takes into account the tissue specific genome-wide MAR association and the potential role of these MARs in developmentally regulated gene expression. The current study lays a foundation to understand the genome wide regulation of chromatin during development.
... Some past studies suggested that the increase of transgene expression by MAR was copy number-or position-independent. 33,34 However, other studies showed that the activity of MARs was not sufficient to confer copy-number-dependent transgene expression. 35,36 In this study, the results showed that a MAR consensus sequence can improve transgene copy number to some extent. ...
Matrix attachment regions (MARs) can enhance transgene expression levels and maintain stability. However, the consensus sequence from MARs and its functional analysis remains to be examined. Here, we assessed a possible consensus sequence from MARs and assessed its activity in stably transfected Chinese hamster ovary (CHO) cells. First, we analyzed the effects of 10 MARs on transfected CHO cells and then analyzed the consensus motifs from these MARs using a bioinformatics method. The consensus sequence was synthesized and cloned upstream or downstream of the eukaryotic vector. The constructs were transfected into CHO cells and the expression levels and stability of enhanced green fluorescent protein were detected by flow cytometry. The results indicated that eight of the ten MARs increased transgene expression in transfected CHO cells. Three consensus motifs were found after bioinformatics analyses. The consensus sequence tandemly enhanced transgene expression when it was inserted into the eukaryotic expression vector; the effect of the addition upstream was stronger than that downstream. Thus, we found a MAR consensus sequence that may regulate the MAR‐mediated increase in transgene expression. Assessed the possible consensus sequence from matrix attachment regions (MARs) and assessed its activity in stably transfected Chinese hamster ovary cells. First found the MAR consensus sequence that may mediate the MAR activity of increasing transgene expression. The levels of transgene expression were not only copy‐number‐dependent, other mechanism may be involved.
... For example, S/MARs from human showed the same insulating effect on transgene expression in other organisms such as Drosophila melanogaster. Other research has shown that a κ intronic S/MAR can be replaced by another S/MAR from genomic location yet still show the same methylation pattern and normal gene expression (Namciu et al., 1998). These findings suggest that the same S/MARs may be applicable across multiple cell types, species and genes. ...
... In agreement with the previously reported characteristics of S/MARs (Girod et al., 2005), we found very few that lay within coding sequences (through the BLASTX analysis). This agrees with the supposed role of S/MARs in creating gene-containing chromatin domains to either facilitate or repress transcription (Namciu et al., 1998;Ma et al., 1999). ...
Binding of intergenic Scaffold/Matrix Attachment Regions (S/MARs) to nuclear matrix proteins is believed to poise adjacent genes for transcription by forming chromatin loops. Vector constructs containing Scaffold/Matrix Attachment Regions (S/MAR) flanking the gene of interest, therefore, are able to enhance recombinant protein expression in mammalian cells. We compared two methods that are based on buffers containing 2M NaCl and Lithium-3,5-diidosalicylate (LIS) to isolate S/MARs from HEK293 and CHO DG44 cell lines. Isolated S/MARs were sequenced using the Illumina HiSeq platform and mapped against CHO DG44 genome contigs and the human genome GRCh37.p13 respectively (Sequence raw data from this article have been deposited at the EMBL Data Libraries under Study ID PRJEB26090 (ERP108063)). The 2M NaCl method produced 16 million S/MAR consensus sequences which included nine million and seven million from HEK293 and CHO DG44 respectively. LIS method, on the other hand, generated thirteen million S/MAR consensus containing 8.4 million and 4.7 million from HEK293 and CHO DG44, respectively. In order to compare all sets of S/MAR consensus, BLASTN analyses were performed based on exact matches. The number of perfect matches between S/MAR sequences produced by both methods was quite low: 0.46% and 0.07% for HEK293 and CHO DG44 cells respectively, indicating that the two methods isolate different sets of S/MARs. Comparison between the two cell lines found six S/MARs in common, with average coverage of 82%, obtained by the 2M NaCl method, but none of these are intergenic. The LIS method gave 38 S/MARs with average coverage of 85%, common to both cell types; of these, 13 were intergenic. We hypothesize that S/MARs from HEK293 and CHO DG44 isolated using the LIS method have the potential to be universal vector expression elements that can overcome the problem of low production yield. © 2018 Nur Shazwani Mohd Pilus, Azrin Ahmad and Nurul Yuziana Mohd Yusof.
... It has been reported that MAR elements enhance transgene expression by facilitating copy number-dependent or position-independent expression [36][37][38][39]. However, some reports have indicated that the activity of MARs is not sufficient to confer copy numberdependent transgene expression in animal systems [40]. ...
Low-level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR-6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR-6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP-B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP-B, DHFR intron MAR element and MAR-6. Additionally, as expected, the three MAR-containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non-MAR-containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP-B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.
... MARs are DNA elements that are part of the nuclear matrix, and are known to reduce what is a known as "position effect variegation" (PEV). PEV is characterized by heterogeneous expression (and in some cases silencing) of randomly integrated transgenes [22][23][24]. The use of MARs has been described in multiple species including swine [25,26]. ...
Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model.
... Chromosomal position effects can be attenuated by flanking genes of interest (for example, transgenes) with insulators to block the effects of neighbouring enhancers and silencers as well as encroaching heterochromatin (Bell et al., 2001). A number of different DNA sequences with insulating activity have been identified in both invertebrate and vertebrate species, including specialized chromatin structures, a portion of the gypsy retrotransposon from the fruit fly, Drosophila melanogaster, sites in the sea urchin histone H3 genes silencing nucleoprotein structure, human matrix attachment regions, the chicken b-globin genes chicken hypersensitive site-4 element, the Xenopus ribosomal RNA genes, the human T-cell receptor-a/d locus and the CCCTC-binding factor (CTCF) factor (Udvardy et al., 1985;Geyer & Corces, 1992;Chung et al., 1993;Palla et al., 1997;Robinett et al., 1997;Zhong & Krangel, 1997;Namciu et al., 1998;Moon et al., 2005). ...
DNA insulators organize independent gene regulatory domains and can regulate interactions amongst promoter and enhancer elements. They have the potential to be important in genome enhancing and editing technologies because they can mitigate chromosomal position effects on transgenes. The orthologous genes of the Anopheles stephensi putative gypsy-like insulator protein complex were identified and expression characteristics studied. These genes encode polypeptides with all the expected protein domains (Cysteine 2 Histidine 2 (C2H2) zinc fingers and/or a bric-a-brac/poxvirus and zinc finger). The mosquito gypsy transcripts are expressed constitutively and are upregulated in ovaries of blood-fed females. We have uncovered significant experimental evidence that the gypsy insulator protein complex is widespread in vector mosquitoes.
... CTCF is also required for proper transcriptional activation at this locus, likely supporting secondary chromatin structure or enhancer function, as the SATB1 and CTCF binding sites do not overlap (Ribeiro de Almeida et al. 2009). Human S/MARs are able to protect transgenes from heterochromatic silencing in Drosophila, suggesting that S/MARs are able to support insulator function (Namciu et al. 1998). ...
Recently, many advancements in genome-wide chromatin topology and nuclear architecture have unveiled the complex and hidden world of the nucleus, where chromatin is organized into discrete neighbourhoods with coordinated gene expression. This includes the active and inactive X chromosomes. Using X chromosome inactivation as a working model, we utilized publicly available datasets together with a literature review to gain insight into topologically associated domains, lamin-associated domains, nucleolar-associating domains, scaffold/matrix attachment regions, and nucleoporin-associated chromatin and their role in regulating monoallelic expression. Furthermore, we comprehensively review for the first time the role of chromatin topology and nuclear architecture in the regulation of genomic imprinting. We propose that chromatin topology and nuclear architecture are important regulatory mechanisms for directing gene expression within imprinted domains. Furthermore, we predict that dynamic changes in chromatin topology and nuclear architecture play roles in tissue-specific imprint domain regulation during early development and differentiation.
... As both S/MARs and insulators can participate in the formation of chromatin domains, their interrelation has been investigated. It was reported that the same DNA fragments, at least in some cases, display both the S/MARs and insulator properties [72,[77][78][79][80][81]. However, it was also demonstrated that these activities can be separated [82] or are only observed for certain genetic constructs [83]. ...
... According to current reports, MARs have been used as an alternative for protection against position effects in vertebrates. They are even more successful in plants and alga [1,3,5,10,11,13,15,16,20,21]. In addition, DNA replication, transcription, repair, splicing, and recombination seem to occur in the nuclear matrix [2, 7 -9], MARs may coexist with core origin replication (ORI), and another fraction may coexist with transcriptional enhancers. ...
In our previous study, the sequence of a matrix attachment region binding protein (MBP) cDNA was cloned from the unicellular green alga Dunaliella salina. However, the nucleotide sequence of this gene has not been reported so far. In this paper, the nucleotide sequence of MBP was cloned and characterized, and its gene copy number was determined. The MBP nucleotide sequence is 5641 bp long, and interrupted by 12 introns ranging from 132 bp to 562 bp. All the introns in the D. salina MBP gene have orthodox splice sites, exhibiting GT at the 5' end and AG at the 3' end. Southern blot analysis showed that MBP only has one copy in the D. salina genome. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
... The most favorable locus for introducing MAR is proposed to be upstream of promoter and enhancer region in expression vector [9]. Even the proper mechanisms by which MAR elements contribute to increase protein being expressed still remain unclear, several studies have reported on the enhancement of transgene expression with copy number-dependent or positionindependent properties of MAR elements [7,14,15]. Recent finding has reported that combination of MAR element with mammalian replication initiation region in CHO DG44 cells significantly initiated gene amplification effectively and spontaneously increased gene copy number [16]. ...
The emergence of monoclonal antibody specific to human growth factor receptor marked the greatest achievement in human fight against cancer. By utilizing a specific antibody, cancer treatment nowadays is getting more specific and efficient. Despite its achievement, the main drawback of antibody therapy remains at its production level. Low yield and transient expression of antibody by mammalian host cells are among the problems discovered during antibody production. Many strategies have been implied to increase yield and stability of protein including improvement of vector expression system with DNA elements integration as well as manipulating the bioreactor environment to increase cell density and attain more products. Our study is currently focused on constructing a high expression vector system using matrix attachment region (MAR) element in targeted therapeutic monoclonal antibody production. In this study, a nuclear halo formation has been successfully optimized for CHO cells. A minimum time of 8 minutes is required for maximum nuclear halo formation, thus exposing the most significant sites containing MAR element in CHO genome. This preliminary step is crucial due to the fact that the nuclear halo formation and MAR elements isolation are specific for different types of cells. Subsequent works are currently being carried out to isolate the MAR element from CHO cells based on the time determined from nuclear halo formation. This work is going to be distinctive from previous studies as the MAR element will be predicted from the host cells genome. The mechanism of which the integrated functional MAR element can increase the antibody production even at random position is through up-regulation of gene transcription by adopting a DNA loop structure of nucleosomes that open the structure for specific transcription factor binding.
