Novel short synthetic matrix attachment region for enhancing transgenic expression in recombinant Chinese hamster ovary cells

Matrix attachment regions (MARs) are DNA fragments with specific motifs that enhance transgenic expression; however, the characteristics and functions of these elements remain unclear. In this study, we designed and synthesized three short chimeric MARs, namely, SM4, SM5, and SM6, with different numbers and orders of motifs on the basis of the features and motifs of previously reported MARs, namely, SM1, SM2, and SM3, respectively. Expression vectors with six synthetic MARs flanking the down or upstream of the expression cassette for enhanced green fluorescence protein (EGFP) were constructed and introduced into Chinese hamster ovary (CHO) cells. Results indicated that the EGFP expression of the CHO cells with transfection bySM4, SM5, or SM6-containing vectors was higher than that of those containing SM1, SM2, or SM3 regardless of the MAR insertion position. The improving effect of SM5 was particularly pronounced. Transgenic expression was further enhanced with the increasing SM5 copy number. Bioinformatics analysis indicated that several arrangements of the DNA-binding motifs for CEBP, FAST, Hox, glutathione, and NMP4 may help increase transgenic expression levels and the average population of highly expressed cells. Our findings on novel synthetic MARs will help establish stable expression systems in mammalian cells.     

Chromosomal location and expression of the single-copy gene encoding high-mobility-group protein HMG-I/Y in Arabidopsis thaliana

A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana has been isolated and characterised by nucleotide sequencing. The 903 bp cDNA contains a 612 bp open reading frame encoding a protein of 204 amino acid residues showing homology to HMG-I/Y proteins from other plant species. The protein contains four copies of the AT-hook motif which is involved in binding A/T-rich DNA. Southern blotting showed that the HMG-I/Y gene was present in a single copy in the Arabidopsis genome. The gene was localised to the top of chromosome 1 by RFLP analysis of F₈ recombinant inbred lines. Northern blotting showed that the gene was expressed in all organs examined, with the highest expression in flowers and developing siliques.     

核基质结合序列（MAR）与基因表达调控

核基质结合序列（MAR）是能在体外与核基质特异结合的DNA序列,广泛存在于染色质Loop结构的边界序列中。随着研究的深入,发现MAR序列不仅在染色质折叠中起到重要作用,影响邻近内源基因表达,而且将MAR序列构建到外源基因表达盒两侧转化动植物时,也影响外源基因的表达。因此,MAR序列是基因组中一种重要的基因间边界序列,为阐明非编码序列在基因表达中的作用和构建真核生物高效表达载体提供了新途径。     

Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells

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.     

The role of laminin in attachment,growth, and differentiation of cultured cells: a brief review

Laminin, a major structural multidomain protein of basement membranes, has been shown to exert a variety of biological activities. Prominent among those are mediation of cell attachment as well as influences on cellular proliferation, differentiation and motility. Distinct domains of laminin have been identified which carry these activities. The active sites on laminin are recognized by cellular receptors, several of which belong to the integrin class of heterodimeric transmembrane proteins. These are in direct contact with intracellular proteins and mediate signals from the extracellular matrix to the cytoskeleton and possibly to other intracellular regulatory systems. The biological activities of laminin may be used to design optimal conditions for the expression of a differentiated phenotype of cells in culture.     

Decellularized matrix of adipose-derived mesenchymal stromal cells enhanced retinal progenitor cell proliferation via the Akt/Erk pathway and neuronal differentiation

Background aims

Retinal progenitor cells (RPCs) are a promising cell therapy treatment for retinal degenerative diseases. However, problems with limited proliferation ability and differentiation preference toward glia rather than neurons restrict the clinical application of these RPCs. The extracellular matrix (ECM) has been recognized to provide an appropriate microenvironment to support stem cell adhesion and direct cell behaviors, such as self-renewal and differentiation.

The epicenter of chromosomal fragility of Fra14A2, the mouse ortholog of human FRA3B common fragile site,is largely attached to the nuclear matrix in lymphocytes but not in other cell types that do not express such a fragility

Common fragile sites (CFSs) correspond to chromosomal regions susceptible to present breaks, discontinuities or constrictions in metaphase chromosomes from cells subjected to replication stress. They are considered as genomic regions intrinsically difficult to replicate and they are evolutionary conserved at least in mammals. However, the recent discovery that CFSs are cell-type specific indicates that DNA sequence by itself cannot account for CFS instability. Nevertheless, the large gene FHIT that includes FRA3B, the most highly expressed CFS in human lymphocytes, is commonly deleted in a variety of tumors suggesting a tumor suppressor role for its product. Here, we report that the epicenter of fragility of Fra14A2/Fhit, the mouse ortholog of human FRA3B/FHIT that like its human counterpart is the most highly expressed CFS in mouse lymphocytes, is largely attached to the nuclear matrix compartment in naive B lymphocytes but not in primary hepatocytes or cortical neurons that do not express such a CFS. Our results suggest a structural explanation for the difficult-to-replicate nature of such a region and so for its common fragility in lymphocytes, that is independent of the possible tumor suppressor role of the gene harboring such CFS.     

Nuclear architecture in the light of gene expression and cell differentiation studies

It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their 'epigenomes'. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.     

Breakpoints of t(4;11) translocations in the human MLL and AF4 genes in ALL patients are preferentially clustered outside of high-affinity matrix attachment regions.

Chromosomal translocations t(4;11) are based on illegitimate recombinations between the human MLL and AF4 genes, and are associated with high-risk acute leukemias of infants and young children. Here, the question was asked, whether a correlation exists between the location of translocation breakpoints within both genes and the location of S/MARs. In "halo mapping experiments" (to define SARs), about 20 kb of MLL DNA was found to be attached to the nuclear matrix. Similar experiments performed for the translocation partner gene AF4 revealed that SARs are spanning nearly the complete breakpoint cluster region of the AF4 gene. By using short DNA fragments in "scaffold reassociation experiments" (to define MARs), similar results were obtained for both genes. However, Distamycin A competition experiments in combination with "scaffold reassociation experiments" revealed specific differences in the affinity of each tested DNA fragment to bind the isolated nuclear matrix proteins. When the latter data were compared with the known location of chromosomal breakpoints for both genes, an unexpected correlation was observed. DNA areas with strong MAR affinity contained fewer translocation breakpoints, while areas with weak or absent MAR affinity showed a higher density of chromosomal breakpoints.     

Activation of Na+ /H+ exchange on rat preadipocyte plasma membrane and its role in cell proliferation and differentiation

An in vitro cultured rat perirenal preadipocyte (PA) was established as a model system to investigate the role of the intracellular pH (pHi) and of the Na~ /H~ exchanger during PA proliferation and differentiation, pH sensitive probe, 2' ,7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), was employed to measure the pHi of PA and to determine the Na~ /H~ exchange activity. The results showed that there was Na~ /H~ exchange activity in the plasma membrane of PA, FCS stimulated DNA synthesis measured by ~3H-TdR incorporation, and the activation of Na~ /H~ exchanger resulted in phi increase (nearly 0.2 pH unit) within 2 min. Ethyl-isopropyl-amiloride (EIPA), a specific Na~ /H~ exchange inhibitor, inhibited Na~ /H~ exchange activity and DNA synthesis. In the absence of serum insulin did not stimulate DNA synthesis but did induce PA differentiation characterized by the appearance of adiposome in the cell and the enhancement of glycerol-3-phosphate dehydrogenase (G_3PDHase) activity. Meantime, insu     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

M-CSF上调cyclinD1/D3和CDK2/6的表达促进HeLa细胞增殖

非分泌型巨噬细胞集落刺激因子(M-CSF)的表达在肿瘤的发生发展过程中发挥重要作用,为探讨胞质M-CSF对细胞增殖的影响,采用基因重组技术构建胞内稳定表达M-CSF的HeLa细胞系,以空载体(pCMV/myc/cyto)转染HeLa细胞和未转染HeLa细胞作为对照,MTT法及反义寡核苷酸抑制实验分析M-CSF对细胞增殖的影响,并计算细胞倍增时间,RT-PCR观察胞内M-CSF对G1期细胞周期相关蛋白的影响．结果显示,与对照组比较,转染M-CSF的HeLa细胞倍增时间明显缩短、增殖能力显著增强,M-CSF的特异性反义寡核苷酸能抑制转染M-CSF的HeLa细胞的增殖,且抑制率随着反义寡核苷酸浓度的增高而增强,转染M-CSF 的HeLa细胞的cyclinD1/D3和CDK2/6 mRNA表达显著升高(P < 0.05)．提示：M-CSF可上调cyclinD1/D3和CDK2/6的mRNA表达,促进HeLa细胞的增殖．     

Perchloric acid-soluble protein regulates cell proliferation and differentiation in the spinal cord of chick embryos

The role of perchloric acid-soluble protein (PSP) was investigated in chick embryos. Fluorescently labeled anti-chick liver (CL)-PSP IgG was injected into the yolk sac in ovo at embryonic day 3, and became localized in neuroepithelial cells. Within 12 h, morphological changes were observed in 37.5% of anti-CL-PSP IgG-injected embryos, and the neuroepithelial cells formed a wavy line. No significant changes were observed in embryos injected with non-immune IgG or PBS. Increased expression of PCNA and decreased expression of neuronal class III beta-tubulin were observed in the spinal cord after anti-CL-PSP IgG injection. These results suggest that PSP controls the proliferation and differentiation of neuroepithelial cells in chick embryos.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells

This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR–survivin promoter–CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC + GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells.     

Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize <Emphasis Type="Italic">adh-1</Emphasis> matrix attachment regions: tissue and developmental specificity in maize transgenic plants

Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms. In plants, contradictory data question the role of MAR sequences. To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region. The MARs have been cloned either 5 to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain. Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny. Furthermore, MARs systematically induced variegation. We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.Abbreviations adh-1 Alcohol dehydrogenase 1 - GUS -Glucuronidase - HSC80 Heat shock cognate 80 gene - MAR Matrix attachment regions - Rsyn-7 Root specific synthetic promoter