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Cmv and sv40

Qin Li, Xiao-Yin Wang, Chun-Peng Zhao, Zheng-Wei Tian, Dan-Hua Xu, Tian-Yun Wang, Jun-He Zhang
OBJECTIVE: To analyze the effects of different promoters and matrix attachment region (MAR) on the expression of transgene in Chinese hamster ovary (CHO) cells. METHODS: The expression vector was constructed by the combination of beta globin MAR (gMAR) with the human cytomegalovirus immediate-early promoter (CMV-IE) and simian virus 40 (SV40) promoter. These vectors were transfected into CHO cells,after 48 h,the transient expression of enhanced green fluorescent protein (eGFP) was observed; G418 was used to screen stably transformed cell lines,and the expression level of eGFP in CHO cells was analyzed by flow cytometry...
January 2018: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
Xiaojun Yu, Wei Geng, Hongchuan Zhao, Guobin Wang, Yijun Zhao, Zhe Zhu, Xiaoping Geng
BACKGROUND Vectors are widely used to drive gene expression using a promoter. However, not all promoters are able to drive ectopic gene expression efficiently, including CMV promoter. Here, we report our data using CMV promoter for high-level gene expression in a B lymphoma cell line DG75. MATERIAL AND METHODS A plasmid (pcDNA3.1(+)) containing the CD21 gene driven under CMV promoter was constructed. The plasmid was stably transfected into a human B lymphoma cell line DG75 for cellular surface CD21 expression, and flow cytometry was used to monitor CD21 expression...
December 15, 2017: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
Youssef Yakkioui, Ernst-Jan M Speel, Jacobus J Van Overbeeke, Merel J M Boderie, Sreedhar Pujari, Axel Zur Hausen, Petra F G Wolffs, Yasin Temel
BACKGROUND: Chordomas are rare tumors assumed to derive from notochordal remnants. We believe that a molecular switch is responsible for their malignant behavior. The involvement of oncogenic viruses has not been studied, however. Thus, in the present study, we investigated the presence of oncogenic viruses in chordomas. METHODS: DNA and RNA from snap-frozen chordoma (n = 18) and chondrosarcoma (n = 15) specimens were isolated. Real-time PCR or RT-PCR was performed to assess the presence of multiple oncogenic viruses, including herpesviridea (herpes simplex virus [HSV]-1, HSV-2, Epstein-Barr virus [EBV], cytomegalovirus, human herpesvirus [HHV]- 6, HHV-7, and Kaposi's sarcoma-associated herpesvirus), polyomaviridea (parvovirus B19 [PVB19], BK virus, JC virus, Simian virus 40, Merkel cell polyomavirus, human polyomavirus [HPyV]-6, and HPyV-7), papillomaviridae, and respiratory viruses...
November 28, 2017: World Neurosurgery
Alexander Raup, Valérie Jérôme, Ruth Freitag, Christopher V Synatschke, Axel H E Müller
Non-viral transfection protocols are typically optimized using standard cells and reporter proteins, potentially underestimating cellular or transgene effects. Here such effects were studied for two human (Jurkat, HEK-293) and two rodent (CHO-K1, L929) cell lines and three fluorescent reporter proteins. Expression of the enhanced green fluorescent protein (EGFP) was studied under the control of the human elongation factor 1 alpha promoter and three viral promoters (SV40, SV40/enhancer, CMV), that of ZsYellow1 (yellow fluorescence) and mCherry (red fluorescence) for the CMV promoter...
September 2016: Biotechnology Reports
Chun-Peng Zhao, Xiao Guo, Si-Jia Chen, Chang-Zheng Li, Yun Yang, Jun-He Zhang, Shao-Nan Chen, Yan-Long Jia, Tian-Yun Wang
Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter...
February 20, 2017: Scientific Reports
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
Pratigya Gautam, Asha Recino, Robert D Foale, 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. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells...
October 2016: Journal of Gene Medicine
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
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
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
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
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
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
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
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
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...
February 9, 2015: Journal of Visualized Experiments: JoVE
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
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
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
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
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