Ebna 1 expression vector software

Whether the oncogenic potential of ebna1 is related to induction of genomic instability, however, has not been addressed. The gb1b2 expression plasmid was constructed by inserting ebna1 amino acids 189 encoding dna into bspei and bgliicut gb2 expression plasmid. A protein pivotal to latent infection by epsteinbarr. Aid 588762 late stage assay provider results for the probe. Expression vector pdef38, introduced by these authors, differs significantly from the widely employed vectors with the core promoter of the human ortholog elongation factor 1 alpha gene ef1a. Ebna1 dependent longterm gene expression in neurons. Autorepression of epsteinbarr virus nuclear antigen 1. Development of expression vectors for escherichia coli based on the pcr2 replicon.

Epsteinbarr virus essential antigen ebna3c attenuates h2ax expression article pdf available in journal of virology 887 january 2014 with 96 reads how we measure reads. If you have already purchased a full version of expression design 4, you do not need to install this free version. Left panel ebvnegative louckes bl cells were cotransfected with a balcv or rhlcv qp reporter plasmid and either an empty expression vector psg5 or an ebna 1 expression vector psg. Maintenance of epsteinbarr virus ebv oripbased episomes. In fact, these vectors contain not only the erebna2 open reading frame. Recombinant ebna 1 protein rebna 1 was isolated from this baculovirus expression vector according to lori frappier personal communication and modifications of frappier and odonnell. Longterm transgene expression and inhibition of hiv1. Longterm transgene expression and allied academies. Creloxp recombination, ebna 1 orip, gene therapy, hiv 1, ribozyme introduction in the application of therapeutics to hiv 1 infection. Enhancement of transgene expression by cotransfection of orip. The vector contains ebv orip and ebna to ensure replication as episomes in human or simian cells, even if the cell line is not ebna positive. Cloning system and protein expression vectors functional protein analysis usually requires recombinant expression of the protein of interest. In addition, the plasmid carries the two essential ebv elements orip and. The choice of the best expression vector depends on the characteristics of the protein.

Epsteinbarr nuclear antigen 1, or ebna1, is required for the replication of the ebv genome as an extrachromosomal element and is a key transcriptional regulator of this viruss latent gene. An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a. Pdf autorepression of epsteinbarr virus nuclear antigen 1. A specific viral gene expression program, known as latency iii 3, is activated. Efficient selection for highexpression transfectants with a novel eukaryotic vector. Improved gene expression pattern using epsteinbarr virus ebvbased plasmid and cationic emulsion.

It comprises a perfectly matched hek293e cell line, cell culture media and expression vectors to provide high protein yields without further optimization. Longterm transgene expression and inhibition of hiv 1 replication by a creloxp ebna 1 orip hiv 1 dependent ribozyme vector. For this purpose, the protein coding sequence is cloned into a suitable expression vector and transferred into cells. Junichi miyazaki of osaka university graduate school of medicine.

The vector according to claim 1, wherein said vector comprises a polynucleotide sequence encoding a selectable marker for expression and growth advantage in a metazoan cell. Geneious prime is connected to plasmapper which allows you to automatically highlight plasmids with common promoters, terminators, cloning sites, restriction sites, reporter genes, affinity tags, selectable marker genes, replication origins and open reading frames. Regulated expression of a transgene introduced on an orip. Construction of epsteinbarr virusbased expression vector. The expression level of ebna 1 antigen appeared to be a limiting factor of the episomebased reprogramming since the introduction of additional vector carrying ebna 1 under control of strong promoter enhanced the ipscforming efficiency. Epsteinbarr nuclear antigen 1 ebna1 is a multifunctional, dimeric viral protein associated. Currently the vectors require ebna 1 expression in trans for episomal function but we are generating new vectors which express ebna 1 from an open reading frame in cis. Late stage assay provider results for the probe development effort to identify inhibitors of the epsteinbarr virus nuclear antigen 1 ebna 1. Furthermore, the combination of a repressor and an activator acting through the tetoperon prevents expression in the offstate and allows precise titration of the expression level of the respective microrna. The vector system employed here is episomal and therefore microrna expression levels are not dependent on the site of integration. The invention utilises the large t antigen and origin of replication of polyoma virus and the ebv nuclear antigen 1 ebna 1 and ebv origin of replication from ebv. Ebna1 is typically expressed in most forms of ebvpositive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Bioassay record aid 588762 submitted by the scripps research institute molecular screening center. Oct 16, 2009 the ebna 1 gene was obtained as a spei fragment from the vector pebac160 23 kindly provided by dr.

In comparison to other eukaryotic expression systemsthe hek. Both vectors contain the ebna 1 gene for episomal expression in primate and canine cell lines. Repression of epsteinbarr virus ebna 1 gene transcription by prb during restricted latency. Get sustained, nonintegrating gene expression in vitro and in vivo with eev episomal expression vectors. Epsteinbarr virus nuclear antigen ebna 3a induces the expression of and interacts with a subset of chaperones and cochaperones. Epsteinbarr nuclear antigen 1 ebna1 has been shown to activate. The intensity of each protein band was estimated using imagequant version 5. Expression vectors overview cloning iba lifesciences.

Ebna1 regulates cellular gene expression by binding cellular. Here we demonstrate that the prts 1 vector can be used to conditionally regulate the expression of micrornas mediating rnainterference, thereby providing a convenient tool to determine gene. Episomal expression vector for metazoan cells university of. Epsteinbarr virus ebvencoded nuclear antigen ebna1 is thought. Analysis of the transfectants indicated that coexpression of ebna 1 and gs enhanced transient expression of a recombinant antibody from a plasmid carrying an orip dna element compared to ebna 1 only transfectants. Extrachromosomal replication by either the more powerful orip cell line independent due to ebna1 gene provided by vector or sv40 ori cell line dependent possible. Lori frappier mcmaster university, ontario, canada. Inducible microrna expression by an allinone episomal. Establishment of an oripebna1based episomal vector transcribing. To address this issue we have profiled the transcriptional. The epsteinbarr virus ebv encoded nuclear antigen ebna 1 regulates virus replication and transcription, and participates in the remodeling of the cellular environment that accompanies ebv induced bcell immortalization and malignant transformation. Analysis of the transfectants indicated that coexpression of ebna1 and gs enhanced transient expression of a recombinant antibody from a plasmid carrying an orip dna element compared to. The p5170 series of vectors represents a first generation of ebv vectors designed to carry genomic dna inserts in human cells.

The introduction of epsteinbarr nuclear antigen 1 ebna 1 can act in trans to increase the expression of cloned genes, is also reported to enhance ips formation 9,19 and correlates with p53. Repression of epsteinbarr virus ebna1 gene transcription. The ebna 1 baculovirus expression vector used in this study was a generous gift from dr. We have compared the transcription profiles of conditional and stable ebna 1 transfected sublines of the ebv negative bcell lymphoma bjab where ebna 1 was expressed for. Automatically annotate plasmid maps and expression vectors. Thus, the switch from lymphoblastoid to a restricted viral program may occur at the sites of a germinal center reaction. Ca00446 and by korea research foundation program year 1997. Longterm stability of large insert genomic dna episomal. Molecular components of airinduced gene expression in hek.

The expression level of ebna 1 antigen appeared to be a limiting factor of the episomebased reprogramming since the introduction of additional vector carrying ebna 1 under control of strong promoter enhanced the ipscforming effi. Epsteinbarr virus essential antigen ebna3c attenuates. Ebna1 has been reported to activate the expression of the recombinase activating genes 1 and 2 rag1 and rag2, and it is therefore conceivable that longterm expression of ebna1 could result in genomic instability as a result of inappropriate expression of the rag genes. Transcription profiling of epsteinbarr virus nuclear antigen. Qmcf cell lines express larget antigen and ebna 1 proteins which bind the viral sequences on the qmcf plasmid and hence support plasmid replication and maintenance in the cells. A novel oripebna1based episomal vector has been constructed that. This is our attempt to examine the effect of ebna 1 on the expression of cellular genes. Plasmids containing orip, the latent replication origin of ebv, can be propagated stably as episomal dna in human cells that express the ebv nuclear antigen 1 ebna1, which binds to orip and functions as the transacting replication initiator. Epsteinbarr virus vectors for gene expression and transfer sclimenti and calos 477 the stable extrachromosomal replication, low mutation rate, and easy establishment of f. Avoid the challenges of viral transduction systems with the new eev nonviral, nonintegrating plasmidbased expression vector system. The latest big news is a publication in science by junying yu et al in the thomson lab, who induced human ips by using oripebna1 plasmid vector 1. Us20110076760a1 viral expression plasmids for production of.

Baculovirus expression vector system bd biosciences. Efficient selection for high expression transfectants with a novel eukaryotic vector. The convenience and ease of editing vector graphics in expression design can be a cornerstone of your windows presentation foundation wpf, microsoft silverlight, and web images. A typical fluorescent images of gfpactin transfected neurons cultured for 23 days 23div with eb or delta ebna vectors upper. Download microsoft expression design 4 free version from. Ebna1s crucial role in viral dna replication, episomal maintenance, and partitioning is well examined whereas its importance for the immortalization process and the tumorgenicity of ebv is. Ebna cells applied to controlled proliferation studies by expression of p27kip1. Due to the identical multiple cloning sites different vector backbones can be easily tested. Prolonged gene expression in mouse lung endothelial cells. Fulllength ebna1 was cloned into the expression vector pcdna3. K1 cell line stably transfected with the polyomavirus py large t lt antigen gene pylt and 2 a dna expression vector, ppyebv encoding the py origin pyori for autonomous plasmid amplification and encoding epstein. The level of hgh in the culture medium was determined in duplicate approximately 40 h. Nonintegrative sustained expression persistent, nonintegrating transgene expression high levels of expression easy to clone formats no special plasmid production nonviral, nonintegrating technology constitutive and inducible vector formats.

Use with snapgene software or the free viewer to visualize additional data and align. Vector configurations tested are shown along the x. Efficient nuclear import and extrachromosomal replication due to vector encoded ebv origin of replication orip and ebna 1 provided by mexi293e cells. Kimnonviral vector for efficient gene transfer to human ovarian. Both vectors contain the ebna1 gene for episomal expression in primate and canine cell lines. Epsteinbarr virus nuclear antigen ebna 3a induces the. Antibodies elicited in response to ebna1 may crossreact. The epsteinbarr virus ebv nuclear antigen 1 ebna1 is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viralassociated tumors. Ebna1, a bifunctional transcriptional activator molecular and.

Gene expression patterns of ebv negative bjab blymphoma. Suspensionadapted chok1 host cells were engineered to express the gene encoding epsteinbarr virus ebv nuclear antigen1 ebna1 with and without the coexpression of the gene for glutamine synthetase gs. Episomal expression vectors eev system biosciences. Panos ioannou, the murdoch childrens research institute and cloned into pe107, resulting in the vector pe108. Pdf carcinomarisk variant of ebna1 deregulates epstein. In the present work, we used cdnamicroarray analysis to study. Both vector formats feature a multiple cloning site mcs to insert a cdna, microrna, etc. We also show that a bac carrying gfp, orip and ebna 1 can be transfected into b16f10 cells with lipofectamine 2000 and can be rescued intact after 5 weeks. Moreover, we inserted the reporter gene egfp to enable visualization of transgene expression figure 1 a. Ppt hekebna expression powerpoint presentation free to. Epsteinbarr virus nuclear antigen 1 activates transcription from. The consistent presence of hhv8 and of ebv in parallel in about 70% of pels suggests an important role of these 2 herpesviruses in the development of this lymphoma. Furthermore, genetic analysis has shown that ebna1 expression is an absolute. Cellbased luciferasebased dose response assay for ebna 1 inhibitors.

Andreas kessell, seeks to apply the wildly successful antibodiesonline business model to world of genomics and transcriptomics research. In order to identify cellular genes regulated by ebna3a expression, cdna encoding ebna3a was incorporated into a recombinant adenoviral vector. Epsteinbarr virus vectors for gene expression and transfer. Jul 17, 2010 ebna 1 is expressed in all ebvassociated malignancies and may play a role in transcription regulation through its dna binding ability. The putative cellular targets of these effects of ebna 1 are largely unknown.

Ebna 1 gene is carried by this plasmid to permit extrachromosomal replication in human, primate, and canine cells. Primer plasmids enzymes tools for genomics scientists. Improved gene expression pattern using epsteinbarr virus. Mechanisms that regulate epsteinbarr virus ebna1 gene. Qmcf technology has several important differences compared to commonly known transient expression and stable cell line expression systems. Cellbased luciferasebased dose response assay for ebna 1. Because they replicate in synchrony with the host cell, they are stably inherited and can be used for longlasting expression up to several monthswithout modifying the host genome. Autorepression of epsteinbarr virus nuclear antigen 1 expression by inhibition of premrna processing. Apr 17, 2003 the development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. W estern blot analysis of flag ip from hela cells with stable expression of flag vector, febna1 b958 or febna1 npc probed with antibody to usp7, tankyrase 1, survivin, or flag.

This method avoids integration of transgenes into the genome, thus reducing the risk of causing mutations. Cancer center, the program in molecular biology and biotechnology, and. Pdf mechanisms that regulate epsteinbarr virus ebna1. The vector according to claim 1, wherein said vector comprises an origin of replication for episomal maintenance in a prokaryotic cell. When an epsteinbarr virus ebv nuclear antigen 1 ebna1 expression vector, pcmvtrebna1, was cotransfected with an origin of latent viral dna replication oripharboring plasmid, poripcmvluciferase, luciferase gene expression was up to 20 times greater than in the absence of ebna1. Transient expression for time and cost efficient protein production. Viral nuclear oncoproteins ebna3a and ebna3c are essential for the efficient immortalization of b cells by epsteinbarr virus ebv in vitro and it is assumed that they play an essential role in viral persistence in the human host. The present invention relates to protein expression systems and in particular to rodent cell expression systems utilising polyoma virus and epstein barr virus elements. Moreover, we inserted the reporter gene egfp to enable visualization of transgene expression figure 1a.

Expression of trastuzumab in different vector configurations is shown. Fdne induction inhibited expression from an ebna1dependent orip reporter. Jun 19, 2015 the expression level of ebna 1 antigen appeared to be a limiting factor of the episomebased reprogramming since the introduction of additional vector carrying ebna 1 under control of strong promoter enhanced the ipscforming efficiency. Mammalia expression vectors cloning iba lifesciences.

An ebvbased plasmid containing the two ebv components, origin of replication orip and ebv nuclear antigen 1 ebna 1, was constructed. Definition the expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cellsthe expression vector is a plasmid engineered to introduce a particular gene into the target cell. Because one disadvantage of these vectors is their large sizes, we examined the effect of partial deletion. Development of expression vectors for escherichia coli. The technology is based on the epsteinbarr nuclear antigen1 oripebna1 that has the ability to replicate in synchrony with the host genome by attaching to the host chromatin and replicating with each cell cycle division. These vectors are designed for highlevel, constitutive expression from either the cmv or rsv promoters. Optimization of protein expression in mammalian cells. Aid 588762 late stage assay provider results for the. The available cloning and expression eev vector formats are shown below. Expression vectors for wildtype ebna1 and these six derivatives were. Ebna1 is expressed from the qp promoter during all latency programs. The principle ebna 1 orip based expression in human embryonic kidney 293 cells 293 stably transformed with ebna 1 gene the cell line is available from atcc and, until recently, also from invitrogen 7 why hek. Establishment and applications of epsteinbarr virusbased.

Ebv is a ubiquitous human gammaherpesvirus which preferentially establishes latent infection in viral infected blymphocytes. Choose from over a million free vectors, clipart graphics, vector art images, design templates, and illustrations created by artists worldwide. Applications for hiv 1 gene therapy, takashi nagawa, y. Bv vectors have made them valuable as shuttle vectors to study mutation 1012 and as cloning vectors for establishment and screening of cdna libraries 16. Enhancement of transgene expression by cotransfection of. Oct 01, 2012 completely redesigned to be faster, more intuitive and with a dedicated mac os x version vector nti express software retains the core, trusted tools of vector nti advance software with a. Plasmid maps of vectors, eb vector and ebna lacking eb vector delta ebna.

Epsteinbarr virusderived vector suitable for longterm. In 2016, the team at antibodiesonline set their sights on the next big thing tools for studying the genome and transcriptome led by managing directors dr. Cho, an episomal expression system for recombinant. Generation of human ipscs from cells of fibroblastic and. The pretro vectors allow efficient retrofitting of bacs with g418 resistance, luciferase andor orip ebna 1 using in vivo expression of cre. When an epsteinbarr virus ebv nuclear antigen 1 ebna1 expression vector, pcmvtrebna1, was cotransfected with an origin of latent viral dna. Cn100577808c rodent expression systems utilising polyoma. Sep 28, 2018 expression of trastuzumab in different vector configurations is shown. Dec 12, 2007 transient expression of ebna1 or the ebna1 mutant n. Mar 11, 2020 figure 1 ebna1 dependent longterm gene expression in neurons. Promega offers a wide range of tools to facilitate cloning. This was confirmed with the retransfection of an ebna 1 only cell line with a gs gene. Ebv occurs naturally as two different viral types type 1 and type 2. Epsteinbarr virus ebvbased vectors are extrachromosomal vectors carrying a replicational origin, orip about 2200 bp and a replication initiation factor ebna1 which are sufficient for autonomous replication.