This page is informational only this vector is not available from addgene please contact the manufacturer for further details. Several properties make sv40 a good candidate to be used as a vector for gene therapy. Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as dna vaccines. In addition to commonly used transformed cell lines such as hela, cho, cos7. Sv40 large t antigen is not responsible for the loss of sting. This pcr product was then cloned into the noti and ngomiv sites of the lpcx vector. An optimal vector for gene therapy should be safe, efficient, nonimmunogenic, and available in high titers. The influence of sv40 polya on gene expression of baculovirus.
The psi expression vector contains the simian virus 40 sv40 enhancer and early promoter region. A live vector vaccine is a vaccine that uses a chemically weakened virus to transport pieces of the pathogen in order to stimulate an immune response. This article is an open access publication abstract there has been a resurgence in gene therapy efforts that is partly fueled by the identi. For example, adenoassociated virus is a small dna virus that may infect both resting and dividing cells. We will email to confirm that your organization can accept shipments. Examples of the assays to which this vector system is well suited are presented in the chapter. Sv40 as an effective gene transfer vector in vivo journal of. The sv40 enhancer is composed of multiple functional elements that can compensate for one another winship herr and jennifer clarke cold spring harbor laboratory cold spring harbor, new york 11724 summary we present evidence that the sv40 enhancer consists of three functional units, a, b, and c, each of which.
The principle limitation of sv40derived vectors is the size of the packageable insert. In the baculovirus prototype autographa californica multiple nucleopolyhedrovirus acmnpv, the polyhedrin promoter very late promoter transcribes its gene by a viral rna polymerase therefore there is no supporting. No or low number of colonies on plate with cdna sample 12 c. Sv40 17kt was cloned from cdna derived from 293 cells transfected with the sv40 er plasmid. Polymerase chain reaction pcr to detect sv40 large t antigen tag dna was performed on. To evaluate this system, we produced sv40 containing firefly luciferase luc as a reporter. An aliquot of the sample was subjected to twodimensional gel. A novel role for rax, the cellular activator of pkr, in. Nevertheless, it may be virtually impossible to rule out such connection. Minimal sample processing ie avoid pbmc purification on ficoll. Transient transfection of 293t cells faculty websites.
Overview this sample consists of a simple form containing four distinct fields. Promega prl family of renilla luciferase control reporter. Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious sv40. The functional elements of the vector are the puc origin of replication puc ori for propagation in li, the matrix attached region from the 5. Use text editor or plasmid mapping software to view sequence. A novel bk virusbased episomal vector for expression of. Pdf identification of fam111a as an sv40 host range.
Adenoviral vector design for highlevel transgene expression. Plasmid sequence and snapgene enhanced annotations. Accelio present applied technology created and tested using. Pdf the influence of sv40 polya on gene expression of. The early sv40 enhancerpromoter region prlsv40 and the cmv immediate early enhancerpromoter region prlcmv typically provide highlevel transcription and, therefore, may be less suitable for coreporter applications involving experimental vectors with robust regulatory elements. Sv40 is a small simian virus that has been extensively used as a viral vector in mammalian cells 1, 2, 3, 4. Expression of sv40 lt cterminus increases early and late viral gene levels. Select the appropriate promoter or test all three in your experimental system. More than 30 years of followup data were available. Sv40 was assigned to the family of papovaviridae, an acro. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro.
Sv40 promoter 30373375 neomycin resistance orf 37324526 puc origin of replication 60496727 ampicillin resistance bla orf 68787735 figure 1 circular map and features of the supercos 1 cosmid vector. Recent molecular biology and epidemiological studies suggest that sv40 may be contagiously transmitted in humans by horizontal infection, independently from the earlier administration of sv40contaminated vaccines. First, the host range is limited to certain primate cells. Cho cells transfected with the sv40 promotercontaining vector maintained 68. For stable expression, the psi vector must be cotransfected with an expression vector containing a selectable gene for mammalian cells. Recent molecular biology and epidemiological studies suggest that sv40 may be contagiously transmitted in humans by horizontal infection, independently from the earlier administration of sv40 contaminated vaccines. Fortunately, functions provided by helper dna molecules might help to overcome these problems. Pdf largescale manufacture and characterization of a. The polyomavirus simian virus 40 sv40 is a potent dna tumor virus, and mounting evidence suggests that it is an emergent human pathogen 1, 10, 12, 39, 49, 50, 66, 111, 123. The properties of constitutive promoters within adenoassociated viral aav vectors have not yet been fully characterized. Sv40 based vectors are efficient gene delivery vehicles for a wide spectrum of ex vivo and in vivo targets, including hematopoetic, liver and kidney cells 18,19.
Three renilla vectors with a different promoter sv40, tk or cmv that can be used as a control reporter for dualluciferase assays e6911, e6921, e6931. The dna to be expressed is cloned into the pmt2t or related vector which contains a sv40 origin, adeno major late promoter, the adeno tripartite leader and the va rna i gene, leading to very high level expression of the recombinant protein. Replicationdefective rd recombinant simian virus 40 sv40 based gene delivery vectors hold a great potential for clinical applications because of their presumed nonimmunogenicity and capacity to induce immune tolerance to the transgene products in humans. After ligation of the xhoidigested end of luc cdna into the pbsv. It has been assumed that all polio vaccines were sv40 free in the united states after 1961 and in other countries after 1962.
The pgl4 vectors are engineered with fewer consensus regulatory sequences and a synthetic gene, which has been codon optimized for mammalian expression. Sv40 is a small dna virus that has served as a powerful model system for dissecting fundamental biological processes including, dna replication, transcription, and neoplastic transformation 14. Use these vectors as the control reporter for dualluciferase assays where renilla or nanoluc luciferase are the experimental reporter. An insertion vector possesses at least one unique restriction site into which new dna can be inserted. The first experiments demonstrating the utility of. Sv40 plasmids vectors can be packaged only if their dna is within the range of 3900 to 5300 bp.
Absence of sv40 t antigen detection and identification of the viral proteins in the lv vector. Sv40, we recently conducted a large, longterm retrospective followup study of birth cohorts exposed or unexposed to sv40contaminated poliovirus vaccine strickler et al. This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. The total protein content and control reporter vector methods are. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. Tag is capable of inducing malignant transformation of a variety of cell types. Simian virus 40 sv40 is a monkey virus that was administered to human populations by contaminated vaccines which were produced in sv40 naturally infected monkey cells. Sv40, we recently conducted a large, longterm retrospective followup study of birth cohorts exposed or unexposed to sv40 contaminated poliovirus vaccine strickler et al. The revertant en hancers were also cloned into a human 6globin expres sion vector to assay directly for restored enhancer func tion. Description pmetluc2reporter is a promoter reporter vector that allows the analysis of promoter function.
Pbmc using two different sv40derived viral vectors. The viral genome is a doublestranded supercoiled dna molecule of 5243 bp that is packaged into a capsid containing the viral structural proteins vp1,vp2, and vp3. The resulting recombination reaction is then transformed into. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement contained in this user manual. Minimal sample processing ie avoid pbmc purification on ficoll vector dna extracted and captured efficiently and reliably realtime pcr simple testing format and analysis for surveillance central data collection and analysis raavcmepo vector strong promoter for low vector dose cmv. One of the primary organs affected by sepsis is the lung, presenting as the acute respiratory distress syndrome ards.
Samples are examined by agarose gel electrophoresis, comparing cut and uncut vector. Lentivirus production is influenced by sv40 large tantigen and chromosomal integration of the vector in hek293 cells article pdf available in human gene therapy 2210. The transforming activity of tag is due in large part to its perturbation of the retinoblastoma and p53 tumor suppressor proteins. In this study, aav vectors, in which enhanced gfp expression was directed by one of the six constitutive promoters human. The ease with which high titer sv40 virus stocks can be made and. The tumors investigated included ependymoma, all brain cancers combined, osteosarcoma, and mesothelioma. Transient and stable gene expression in mammalian cells. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement ver. Order oligonucleotide pairs that contain the desired mirna target region and, when annealed and ligated into the pmirglo vector, result in the mirna target region in the correct 5 to 3 orientation.
Adenoassociated virus aav as a vector for gene therapy michael f. A comparative analysis of constitutive promoters located in. They may not be used for any other purpose, including, but. Insertional vectors a large segment of the nonessential region has been deleted, and the two arms ligated together. The p53 tumor suppressor was originally discovered in 1979 as a 53kda simian virus 40 sv40 large t antigen tagassociated protein 92. Sv40 tag is known to bind to and inactivate several tumor suppressor genes, including p53. For example, in the genome of the most extensively studied baculo. The simian virus 40 polyadenylation signal sv40 polya has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems bevs. Although the sv40 vector system offers a rapid and efficient way to introduce and express foreign dna into cells, it also has limitations. A cv1p or u2 os cells were transfected with vector control m, viral dna encoding wild type sv40 wt, host range. Restriction enzymes have proved to be invaluable for the physical mapping of dna. Sep 03, 2014 insertional vectors a large segment of the nonessential region has been deleted, and the two arms ligated together. Sv40 large t antigen is not responsible for the loss of.
Ill site at 5171 just upstream from the initiation codon for the t and t antigens is used as the 5 cloning site and. All three ptracer vectors have the following features. Sv40 intron, a potent strong intron element that effectively. Like other polyomaviruses, sv40 is a dna virus that has the potential to cause tumors in animals, but most often persists as a latent infection.
In conclusion, sv40 intron is a potent strong intron element that increases transgene expression, which can readily be used to more ef. Map and features for pcdf1mcs2ef1copgfp vector 19 d. Pdf lentivirus production is influenced by sv40 large t. To study the association between simian virus 40 sv40 and human hepatocarcinogenesis. Sv40 promoter for highlevel constitutive expression of your gene of interest cmv promoter for highlevel constitutive expression of the cycle 3 gfpzeocin fusion sv40 origin for episomal replication and simple vector rescue in cell lines expressing the sv40 large t antigen the ptracercmv2. Vaccine contamination with sv40 occurred in the united states between 1955 and 1961 simian vacuolating virus 40, known as sv40, is a virus found in monkeys and humans, which has the potential to cause cancer, mainly in animals, although research indicates that it doesnt cause cancer in humans. The sv40 enhancer is composed of multiple functional elements.
There is currently no single vector with all these advantages. The early sv40 enhancerpromoter region prl sv40 and the cmv immediate early enhancerpromoter region prlcmv typically provide highlevel transcription and, therefore, may be less suitable for coreporter applications involving experimental vectors with robust regulatory elements. Generation of a verobased packaging cell line to produce. Sv40 large t antigen simian vacuolating virus 40 tag is a hexamer protein that is a dominantacting oncoprotein derived from the polyomavirus sv40. Use of sv40based vectors to transduce foreign genes to. Recently, the institute of medicine of the national academies concluded that the biological evidence is strong that sv40 is a transforming virus and that the biological evidence is of moderate. For replacement of the early genes, usually a hind. Use of simian virus 40 replication to amplify epsteinbarr virus. This vector can be used for both transient and stable expression of genes.
Emergent human pathogen simian virus 40 and its role in cancer. Second, transfection of most permissive cells with recombinant sv40 vectors culminates in death, limiting studies to transient periods. The sv40 enhancer is composed of multiple functional elements that can compensate for one another. Use with snapgene software or the free viewer to visualize additional data and align other sequences.
Jul 16, 2016 sv40 plasmids vectors can be packaged only if their dna is within the range of 3900 to 5300 bp. Vectors bicistronically linking a gene of interest to the. Immunoblots of the purified viral vectors with antibodies specific for hiv p24 capsid and p17 matrix. Factors influencing the production of recombinant sv40 vectors.
Dec 14, 2015 the influence of sv40 polya on gene expression of baculovirus expression vector systems article pdf available in plos one 1012. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Some oral poliovirus vaccines were contaminated with. Sv40 has been widely studied as a model eukaryotic virus, leading to many early discoveries in eukaryotic dna. The most stable expression was achieved under the sv40 promoter. Clontech products are to be used for research purposes only. The sv40 genome is divided into early and late regions, with the early region coding for the large and small tags and the late region encoding the capsid proteins vp1, vp2, and vp3 fig. However, the clinical use of sv40 vectors has been hampered by the lack of a packaging cell line that produces replicationcompetent. That is, for example rna from svlucinfected cells, as well as rna from mockinfected cells, were. Three ptracer mammalian expression vectors are available that express gfp fused to the selectable marker zeocin. Three renilla reporter vectors used to clone a putative promoter element for proteins with varying half lives or as a control vector in dual. The sv40 late polya signal sequence is positioned downstream of rluc to provide efficient transcription termination and mrna polyadenylation in addition to the modified rluc reporter gene, all prl vectors are isolated from a dam dcm e. Consequently, we devised a gene transfer system based on simian virus40 sv40 1 as a vector. Gene transfer to the rhesus monkey brain using sv40.
Simian virus 40 vectors for pulmonary gene therapy. Svsod1, svgpx1, svrevm10 or a control sv40 vector, svbugt were injected into the caudate nucleus in m. This finding demonstrates that transiently expressed svlt induces replication of its own plasmid, resulting in superenhanced expression due to a positive feedback loop, shown diagrammatically in figure 1b. Sv40 infects a wide range of cell types from humans and other mammals, and expresses its genes in them. The transfer plasmid for the ad5xpgkegfpsv40 vector was constructed by ligating an expression cassette encoding murine pgk1 promoter, egfp gene and sv40. In fact, without restriction enzymes, the biotechnology industry would certainly not have flourished as it has. Adenoassociated virus aav as a vector for gene therapy. This work examines the feasibility of applying simian virus 40 sv40 vectors for. The sv40 enhancer is composed of multiple functional. Since these small genomes do not have much dispensable dna, it is almost impossible to construct a functional vector with any added genes to it. The xhoi sites of the luc cdna and the opened pbsv. This is a free resource for the scientific community that is compiled by addgene. Following a who requirement that was prompted by the detection of sv40 in some human tumors, we conducted a multilaboratory study to test for sv40 polio vaccines prepared.
A single administration of aav vector results in efficient gene transfer in a. Pdf the simian virus 40 polyadenylation signal sv40 polya has been. Replicationdefective rd recombinant simian virus 40 sv40based gene delivery vectors hold a great potential for clinical applications because of their presumed nonimmunogenicity and capacity to induce immune tolerance to the transgene products in humans. Email us at email protected with your order or deposit number so we can prioritize it. After 30 day transfection, the mfi for each vector was measured again, and lower, medium, higher producers % m1, m2, m3 and coefficient of variation cv of each sample were determined at the same time using a facscalibur cytometer.
Sv40 tag dna sequences, present in a small proportion of. The rationale for this approach was based on several important aspects of sv40 virus and its activity. Sv40 is an abbreviation for simian vacuolating virus 40 or simian virus 40, a polyomavirus that is found in both monkeys and humans. Gene transfer to the rhesus monkey brain using sv40derived. List of sequencing primers for cloning vectors and orf. Sepsis remains the leading cause of death in critically ill patients. Using this virus, efficient gene delivery and expression was observed and measured in numerous cell types of human, primate, and rodent origin. Pdf bookmark sample page 1 of 4 pdf bookmark sample sample date. Chapter 6 sv40 virus expression vectors sciencedirect.
Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. The complete sequence and list of restriction sites are available at. For example, published methods for wildtype sv40 production and. Large tag of sv40 strain 776 contains 708 amino acids and is a very multifunctional protein fig.
They offer unparalleled opportunities for diagnosing dna sequence content and are used in fields as disparate as criminal forensics and basic research. The presence of the sv40 ori markedly enhances transient svlt protein expression when compared to the vector lacking this element figure 1a. Simian virus 40 in humans infectious agents and cancer. Each vector uses a different set of promoters to express the gene of interest and the cycle 3 gfpzeocin fusion.