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https://www.readbyqxmd.com/read/27617744/enterovirus-a71-dna-launched-infectious-clone-as-a-robust-reverse-genetic-tool
#1
Chee Wah Tan, Han Kang Tee, Michelle Hui Pheng Lee, I-Ching Sam, Yoke Fun Chan
Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal...
2016: PloS One
https://www.readbyqxmd.com/read/27572655/promoter-optimisation-of-lentiviral-vectors-for-efficient-insulin-gene-expression-in-canine-mesenchymal-stromal-cells-potential-surrogate-beta-cells
#2
Pratigya Gautam, Robert D Foale, Asha Recino, Jing Zhao, Shu Uin Gan, Maja Wallberg, Roy Calne, Andrew M L Lever
BACKGROUND: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. Here we optimised insulin production using lentiviral transduced canine MSCs aiming to evaluate their ability for use as surrogate beta cells...
August 30, 2016: Journal of Gene Medicine
https://www.readbyqxmd.com/read/27226236/impact-of-different-promoters-on-episomal-vectors-harbouring-characteristic-motifs-of-matrix-attachment-regions
#3
Xiao-Yin Wang, Jun-He Zhang, Xi Zhang, Qiu-Li Sun, Chun-Peng Zhao, Tian-Yun Wang
We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and β-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs...
2016: Scientific Reports
https://www.readbyqxmd.com/read/27177910/the-pacific-white-shrimp-%C3%AE-actin-promoter-functional-properties-and-the-potential-application-for-transduction-system-using-recombinant-baculovirus
#4
Yingli Shi, Jianhai Xiang, Guangzhou Zhou, Tetsuzan Benny Ron, Hsin-I Tong, Wen Kang, Si Sun, Yuanan Lu
A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines...
June 2016: Marine Biotechnology
https://www.readbyqxmd.com/read/26883397/quantitative-analyses-of-core-promoters-enable-precise-engineering-of-regulated-gene-expression-in-mammalian-cells
#5
Christopher Ede, Ximin Chen, Meng-Yin Lin, Yvonne Y Chen
Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins...
May 20, 2016: ACS Synthetic Biology
https://www.readbyqxmd.com/read/26620302/evidence-for-transcriptional-interference-in-a-dual-luciferase-reporter-system
#6
Guo-Qing Wu, Xiao Wang, Hong-Ying Zhou, Ke-Qun Chai, Qian Xue, Ai-Hong Zheng, Xiu-Ming Zhu, Jian-Yong Xiao, Xu-Hua Ying, Fu-Wei Wang, Tao Rui, Li-Yun Xu, Yong-Kui Zhang, Yi-Ji Liao, Dan Xie, Li-Qin Lu, Dong-Sheng Huang
The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs...
December 1, 2015: Scientific Reports
https://www.readbyqxmd.com/read/26462050/a-novel-assay-revealed-that-ribonucleotide-reductase-is-functionally-important-for-interstrand-dna-crosslink-repair
#7
Naoaki Fujii, Benjamin J Evison, Marcelo L Actis, Akira Inoue
Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells...
November 1, 2015: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/26262584/intradermal-delivery-of-dna-encoding-hcv-ns3-and-perforin-elicits-robust-cell-mediated-immunity-in-mice-and-pigs
#8
B Grubor-Bauk, W Yu, D Wijesundara, J Gummow, T Garrod, A J Brennan, I Voskoboinik, E J Gowans
Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter...
January 2016: Gene Therapy
https://www.readbyqxmd.com/read/26053031/identification-of-pathogen-signatures-in-prostate-cancer-using-rna-seq
#9
Yunqin Chen, Jia Wei
Infections of the prostate by bacteria, human papillomaviruses, polyomaviruses, xenotropic murine leukemia virus (MLV)-related gammaretroviruses, human cytomegaloviruses and other members of the herpesvirus family have been widely researched. However, many studies have yielded conflicting and controversial results. In this study, we systematically investigated the transcriptomes of human prostate samples for the unique genomic signatures of these pathogens using RNA-seq data from both western and Chinese patients...
2015: PloS One
https://www.readbyqxmd.com/read/25742305/aberrant-splicing-in-transgenes-containing-introns-exons-and-v5-epitopes-lessons-from-developing-an-fshd-mouse-model-expressing-a-d4z4-repeat-with-flanking-genomic-sequences
#10
Eugénie Ansseau, Jacqueline S Domire, Lindsay M Wallace, Jocelyn O Eidahl, Susan M Guckes, Carlee R Giesige, Nettie K Pyne, Alexandra Belayew, Scott Q Harper
The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles...
2015: PloS One
https://www.readbyqxmd.com/read/25742071/generation-of-plasmid-vectors-expressing-flag-tagged-proteins-under-the-regulation-of-human-elongation-factor-1%C3%AE-promoter-using-gibson-assembly
#11
Petar N Grozdanov, Clinton C MacDonald
Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags...
2015: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/25589073/selective-blockade-of-cd28-mediated-t-cell-costimulation-protects-rhesus-monkeys-against-acute-fatal-experimental-autoimmune-encephalomyelitis
#12
Krista G Haanstra, Karin Dijkman, Noun Bashir, Jan Bauer, Caroline Mary, Nicolas Poirier, Paul Baker, Claire L Crossan, Linda Scobie, Bert A 't Hart, Bernard Vanhove
Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0...
February 15, 2015: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/25307181/impact-of-using-different-promoters-and-matrix-attachment-regions-on-recombinant-protein-expression-level-and-stability-in-stably-transfected-cho-cells
#13
Steven C L Ho, Mariati, Jessna H M Yeo, Shiyi Goh Fang, Yuansheng Yang
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...
February 2015: Molecular Biotechnology
https://www.readbyqxmd.com/read/24984206/transcriptional-activity-assessment-of-three-different-promoters-for-mouse-in-utero-electroporation-system
#14
Yanli Liu, Sulei Fu, Rongcheng Niu, Ciqing Yang, Juntang Lin
In utero electroporation (IUE) is a simple and rapid approach to in vivo investigate exogenous gene function in mouse brain, and intensive studies using IUE have greatly contributed to analyze the characterization of specific steps during mouse brain development. Because the efficiency of IUE is highly dependent on the plasmid used and its concentration, and the transcriptional activity of plasmid is not only regulated by the host defense system, but also by the promoter of the expression vectors. Therefore, in the present study, we evaluated the transcriptional activity of three commonly used promoters, CMV, CAG and SV40, in IUE system through measuring the fluorescence intensity of green fluorescent protein which serves as an indicator...
July 2014: Plasmid
https://www.readbyqxmd.com/read/24893109/comparative-transcriptional-activity-of-five-promoters-in-bac-cloned-mdv-for-the-expression-of-the-hemagglutinin-gene-of-h9n2-avian-influenza-virus
#15
COMPARATIVE STUDY
Chengtai Ma, Zhenjie Zhang, Peng Zhao, Luntao Duan, Yaoyao Zhang, Fushou Zhang, Wenqing Chen, Zhizhong Cui
On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters...
September 2014: Journal of Virological Methods
https://www.readbyqxmd.com/read/24714676/characterization-of-constitutive-promoters-for-piggybac-transposon-mediated-stable-transgene-expression-in-mesenchymal-stem-cells-mscs
#16
Sheng Wen, Hongmei Zhang, Yasha Li, Ning Wang, Wenwen Zhang, Ke Yang, Ningning Wu, Xian Chen, Fang Deng, Zhan Liao, Junhui Zhang, Qian Zhang, Zhengjian Yan, Wei Liu, Zhonglin Zhang, Jixing Ye, Youlin Deng, Guolin Zhou, Hue H Luu, Rex C Haydon, Lewis L Shi, Tong-Chuan He, Guanghui Wei
Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells...
2014: PloS One
https://www.readbyqxmd.com/read/24709399/construction-of-recombinant-marek-s-disease-virus-mdv-lacking-the-meq-oncogene-and-co-expressing-aiv-h9n2-ha-and-na-genes-under-control-of-exogenous-promoters
#17
Zhenjie Zhang, Wenqing Chen, Chengtai Ma, Peng Zhao, Luntao Duan, Fushou Zhang, Aijun Sun, Yanpeng Li, Hongqin Su, Sifei Li, He Cui, Zhizhong Cui
To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination...
July 10, 2014: Journal of Biotechnology
https://www.readbyqxmd.com/read/24700644/quantification-of-cytosolic-plasmid-dna-degradation-using-high-throughput-sequencing-implications-for-gene-delivery
#18
Rahul Rattan, Anna U Bielinska, Mark M Banaszak Holl
BACKGROUND: Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. METHODS: Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches. RESULTS: The poly(A) region between the β-lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex-treated cytosol...
March 2014: Journal of Gene Medicine
https://www.readbyqxmd.com/read/24634184/evaluation-of-three-different-promoters-driving-gene-expression-in-developing-chicken-embryo-by-using-in-vivo-electroporation
#19
COMPARATIVE STUDY
C Q Yang, X Y Li, Q Li, S L Fu, H Li, Z K Guo, J T Lin, S T Zhao
To investigate the variance of exogenous gene expression driven by different promoters by in vivo electroporation, 3 plasmid vectors carrying different promoters were selected, and their driving strength was compared in developing chicken embryos. The 3 promoters included: 1) the CAG promoter (containing the cytomegalovirus (CMV) immediate early enhancer and the chicken β-actin promoter), 2) the CMV promoter (the human CMV immediate early region enhancer), and 3) the SV40 promoter (Simian virus 40). The intensity of GFP expression driven by the 3 promoters was detected by fluorescence microscopy...
2014: Genetics and Molecular Research: GMR
https://www.readbyqxmd.com/read/24526517/dramatic-increase-in-expression-of-a-transgene-by-insertion-of-promoters-downstream-of-the-cargo-gene
#20
Masakiyo Sakaguchi, Masami Watanabe, Rie Kinoshita, Haruki Kaku, Hideo Ueki, Junichiro Futami, Hitoshi Murata, Yusuke Inoue, Shun-Ai Li, Peng Huang, Endy Widya Putranto, I Made Winarsa Ruma, Yasutomo Nasu, Hiromi Kumon, Nam-Ho Huh
For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels...
July 2014: Molecular Biotechnology
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