Promoterless gene trap vectors have been widely used for high-efficiency gene targeting and random mutagenesis in embryonic stem (ES) cells. Unfortunately, such vectors are only effective for genes expressed in ES cells and this has prompted the development of expression-independent vectors. These polyadenylation (poly A) trap vectors employ a splice donor to capture an endogenous genes polyadenylation sequence and provide transcript stability. However, the spectrum of mutations generated by these vectors appears largely restricted to the last intron of target loci due to nonsense-mediated mRNA decay (NMD) making them unsuitable for gene targeting applications. Here, we present novel poly A trap vectors that overcome the effect of NMD and also employ RNA instability sequences to improve splicing efficiency. The set of random insertions generated with these vectors show a significantly reduced insertional bias and the vectors can be targeted directly to a 5′ intron. We also show that this ...
Title: Transductional Targeting with Recombinant Adenovirus Vectors. VOLUME: 2 ISSUE: 3. Author(s):Valerie Legrand, Philippe Leissner, Arend Winter, Majid Mehtali and Monika Lusky. Affiliation:CAREXS.A., 11 rue Humann, 67000 Strasbourg, France. Keywords:Adenovirus Vectors, TROPISM, Fiber Protein, Monoclonal antibody. Abstract: Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery ...
TY - JOUR. T1 - Retrograde transfer of replication deficient recombinant adenovirus vector in the central nervous system for tracing studies. AU - Kuo, Hui. AU - Ingram, Donald K.. AU - Crystal, Ronald. AU - Mastrangeli, Andrea. PY - 1995/12/24. Y1 - 1995/12/24. N2 - We assessed the application of a replication deficient recombinant adenovirus vector as a retrograde tracer in neural pathway studies. The adenovirus vector, Ad.RSVBgal, containing the intracellular marker gene, β-galactosidase, was injected directly into the laterodorsal striatum of rats. The retrograde transport of the vector from the injection site was clearly visible in the cerebral cortex, thalamic nucleus, and substantia nigra. No evidence for anterograde transport of the vector was found. When the vector was injected into the genu of the corpus callosum, little uptake of the vector by fibers was noted which suggested that uptake by fibers-of-passage should not be a problem in tracing studies. The present study demonstrates ...
Viral vectors are superior tools for gene therapy and as a genetic vaccine platform because viruses have evolved to efficiently infect and transfer their genomes to cells. Several impressive successes in viral vector-based gene therapies have been reported in humans, including restoration of vision in patients with Lebers congenital amaurosis by retinal gene transfer and cures for severe immune deficiencies by gene transfer to hematopoietic stem cells. However, the mammalian immune system has evolved in parallel to fend off invading pathogens such as viruses. Innate and antigen-specific adaptive immune responses against viral vectors and therapeutic transgene products pose serious hurdles for successful gene therapy. Pre-existing immunity in humans, resulting from prior exposure to the parent virus that forms the basis for the gene transfer vehicle may be derived from, often prevents efficient gene transfer. This problem also reduces our ability to use certain vectors for genetic vaccination or in anti
Oxford Biomedica used Antha to optimize their lentiviral vector transfection/transduction system and improve production efficiency and robustness. This resulted in ~40 hours of time saving, a 3-10-fold increase in vector titre upon transduction and an 81% reduction in pure error.
Vector construction. Construction of recombinant adenoviral vectors expressing human IL12 under control of doxycycline by calcium phosphate-mediated coprecipita
We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green
The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. respectively. The strength of the GFP fluorescence in the body was comparable to the proportion of GFP-positive leukocytes. Moreover, the rate of recurrence of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje Rabbit Polyclonal to PPP2R3B cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques. Introduction The Moloney murine leukemia virus (MoMLV)-based retrovirus vector has been widely used to transfer genes into dividing eukaryotic cells [1]. MoMLV and MoMLV-derived retroviral vectors are not active ...
There is growing interest in the use of lentiviral vectors, particularly for cancer immunotherapy and the treatment of monogenic diseases. Manufacturing of these vectors is challenging primarily due to cytotoxic effects of vector components resulting in low cell culture titres and vector instability leading to low purification yields. In addition, currently used processes are typically not scalable as they rely on adherently cultured cells and unit operations such as batch centrifugation and gel filtration. To improve process scalability, suspension adaptation of a lentiviral vector packaging cell line was attempted, however, cell aggregation could not be prevented. For vector clarification it was found that membranes with pore sizes of 0.22 µm resulted in recoveries less than 50%, whereas the use of 0.45 µm membranes resulted in recoveries close to 100%. Successful vector concentration utilising centrifugal filters was possible with a membrane molecular weight cut-off (MWCO) of 100 kDa, ...
The development of a reverse genetic system has enabled the genetic engineering of negative-strand RNA viruses. This system has been used to analyze the function of viral genes and to construct recombinant viruses which express foreign proteins. In this study, we made an improvement to this system by devising a new method to generate the F-defective SeV vector from a cloned cDNA of a defective RNA genome. This is the first report on constructing a replicon-based RNA vector in the family Paramyxoviridae which replicates in infected cells but does not infect neighboring cells. The improvements achieved in this study are (i) optimization of RNP recovery efficiency by using a UV-inactivated recombinant vaccinia virus expressing T7 RNA polymerase, (ii) construction of an inducible F-expressing packaging LLC-MK2 cell line supplemented with the F protein intrans, and (iii) development of a transfection process for RNP recovered from LLC-MK2 cells. An attempt to recover the F-defective SeV vector ...
TMEM166 is a novel programmed cell death-related molecule. In this report, we constructed a recombinant adenovirus 5-TMEM166 vector (Ad5-TMEM166) and evaluated its expression and anti-tumor activities in vitro and in vivo. Cell viability analysis revealed that the adenovirus-mediated increase of TMEM166 inhibited tumor cell growth in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy (via inhibition of mTOR and activation of p70S6K) and apoptosis (via caspase-3 activation), both of which contributed to cell death and suppression of tumorigenicity. Our data indicated that Ad5-TMEM166 may be a novel gene therapy candidate for cancer. (C) 2012 Elsevier Ireland Ltd. All rights reserved. ...
First-generation, E1-deleted adenoviral vectors (E1-AV) can transduce the vascular endothelium with high efficiency, but their use is limited by the resulting acute endothelial injury and the long-term development of intimal hyperplasia. To reduce the impact of viral proteins on the gene-modified cells, a second-generation adenoviral vector with an additional pair of deletions in the E4 region was developed. To determine whether this E1/E4-AV vector would be useful for vascular gene transfer, we directly compared the efficiency of gene transfer to uninjured rabbit carotid arteries using either an E1/E4-AV or an E1-AV vector encoding beta-galactosidase. Both vectors efficiently transduced vascular endothelium; however, the E1/E4-AV vector gene-modified vessels showed higher beta-galactosidase expression 10 days after gene transfer. Importantly, the E1/E4-AV vector produced substantially less endothelial cell activation, less inflammation, and reduced neointimal hyperplasia compared with the E1-AV vector
Sirion Biotech offers one of the most comprehensive viral vector technology platforms addressing all three major vector types (adenovirus, lentivirus and AAV). Services range from custom virus design to virus vector productions and cell modelling projects, including superior gene knockdown strategies (RNAiONE) and multicistronic expression systems.. ...
Sirion Biotech offers one of the most comprehensive viral vector technology platforms addressing all three major vector types (adenovirus, lentivirus and AAV). Services range from custom virus design to virus vector productions and cell modelling projects, including superior gene knockdown strategies (RNAiONE) and multicistronic expression systems.. ...
Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung.
Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 - 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in
Many viral vector systems have been developed for gene delivery. These include retroviruses, adenoviruses, adenoassociated viruses and herpes simplex virus. Retrovirus vector system: Replication defective retrovirus vectors that are harmless are being used. A plasmid in association with a retrovirus, a therapeutic gene and a promoter is referred to as plasmovirus. The plasmovirus is capable of carrying a DNA (therapeutic gene) of size less than 3.4 kb. Replication defective virus particles can be produced from the plasmovirus. As such, for the delivery of genes by retroviral vectors, the target cells must be in a dividing stage. But majority of the body cells are quiescent. In recent years, viral vectors have been engineered to infect non-dividing cells. Further, attempts are on to include a DNA in the retroviral vectors (by engineering env gene) that encodes for cell receptor protein. If this is successfully achieved, the retroviral vector will specifically infect the target tissues. Adenoviral ...
The researchers tested the new vectors in mice and monkeys and compared the results to reverse-oriented vectors. They found that the new vectors could transfer a much higher viral load-up to six times more therapeutic beta-globin genes than the conventional vectors-and had four to 10 times higher transduction efficiency, a measure of the ability to incorporate corrective genes into repopulating bone marrow cells. The new vectors also showed a capacity for longevity, remaining in place four years after transplantation. Researchers also found that they could be produced in much higher amounts than the conventional vectors, potentially saving time and lowering costs associated with large-scale vector production.. "Our lab has been working on improving beta-globin vectors for almost a decade…and finally decided to try something radically different-and it worked," Tisdale said. "These findings bring us closer to a curative gene therapy approach for hemoglobin disorders.". The new vector, for which ...
Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreichs ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green
OBJECTIVE: Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. METHODS: Oversized (|8kb) transgene constructs containingABCA4coding sequence were packaged as truncated fragments |5kb in size into various AAV serotypes.In vitrotransductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. RESULTS: Transductions with fAAV vector preparations yieldedABCA4mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full lengthABCA4CDS with additional
A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology, bioengineering, and molecular genetics, MCSs let a molecular biologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms (GMOs). One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18. Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster (the polylinker). It has restriction sites for various restriction enzymes, including EcoRI, BamHI and PstI. Another vector used in genetic ...
Vectors based on adenovirus (Ad) are one of the most commonly utilized platforms for gene delivery to cells in molecular biology studies and in gene therapy applications. Ad is also the most popular vector system in human clinical gene therapy trials, largely due to its advantageous characteristics such as high cloning capacity (up to 36 kb), ability to infect a wide variety of cell types and tissues, and relative safety due to it remaining episomal in transduced cells. The latest generation of Ad vectors, helper‑dependent Ad (hdAd), which are devoid of all viral protein coding sequences, can mediate high-level expression of a transgene for years in a variety of species ranging from rodents to non-human primates. Given the importance of histones and chromatin in modulating gene expression within the host cell, it is not surprising that Ad, a nuclear virus, also utilizes these proteins to protect the genome and modulate virus- or vector‑encoded genes. In this review, we will discuss our current
Functional titer is defined as the number of functional vector particles required to infect a cell, present in a volume. There are several methods for measuring titer. Some are more reliable than others. Other methods include: measuring the p24 concentration/ml, measuring RNA equivalents, Transducing units/ml, or measuring mRNA equivalents. The first two are unreliable because they tend to overestimate the vector titer. The amount of p24 measured comes from functional particles, free p24, and nonfunctional vector particles. The RNA assays measure defective particles as well.. Reliable methods are determined by transduction of cells following limiting dilution of vector and subsequent evaluation of reporter protein activity (eGFP) or by the number of colonies following antibiotic selection.. The most straightforward method is to quantify functional vector titer by employing fluorescence and FACs. This is the method employed by the ViraCore. The method does have some limitations including being ...
In molecular biology, subcloning is a technique used to move a particular DNA sequence from a parent vector to a destination vector. Subcloning is not to be confused with molecular cloning, a related technique. Restriction enzymes are used to excise the gene of interest (the insert) from the parent. The insert is purified in order to isolate it from other DNA molecules. A common purification method is gel isolation. The number of copies of the gene is then amplified using polymerase chain reaction (PCR). Simultaneously, the same restriction enzymes are used to digest (cut) the destination. The idea behind using the same restriction enzymes is to create complementary sticky ends, which will facilitate ligation later on. A phosphatase, commonly calf-intestinal alkaline phosphatase (CIAP), is also added to prevent self-ligation of the destination vector. The digested destination vector is isolated/purified. The insert and the destination vector are then mixed together with DNA ligase. A typical ...
There are currently over 417 human clinical trials involving retroviral gene therapy registered in the Journal of Gene Medicine database (http://www.abedia.com/wiley/vectors.php, accessed in July, 2016). The first successful gene therapy protocol occurred in the 1990s. In that protocol, two patients with severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency were treated with a retroviral vector carrying the ADA coding sequence under the transcriptional control of the promoter/enhancers of the long terminal repeat of the MLV. ADA disease is characterized by defective T and natural killer cell maturations as well as low B cell function, causing recurrent infections. In this pioneer trial, one of the treated patients recovered cell counts and function, showing no adverse effects after 4 years. The response was more limited in the second patient primarily due to lower transduction efficacy; however, other causes could have contribute to this low efficiency such as ...
include ,iostream, #include ,cstdlib, #include ,cstring, using namespace std; class Element { private: int number; public: Element() : number(0) { cout ,, "ctor" ,, endl; } Element(int num) : number(num) { cout ,, "ctor" ,, endl; } Element(const Element& e) : number(e.number) { cout ,, "copy ctor" ,, endl; } Element(Element&& e) : number(e.number) { cout ,, "right value ctor" ,, endl; } ~Element() { cout ,, "dtor" ,, endl; } void operator=(const Element& item) { number = item.number; } bool operator==(const Element& item) { return (number == item.number); } void operator()() { cout ,, number; } int GetNumber() { return number; } }; template,typename T, class Vector { private: T* items; int count; public: Vector() : count{ 0 }, items{ nullptr } { } Vector(const Vector& vector) : count{vector.count} { items = static_cast,T*,(malloc(sizeof(T) * count)); memcpy(items, vector.items, sizeof(T) * count); } Vector(Vector&& vector) :count{ vector.count }, items{ vector.items } { vector.items = nullptr; ...
You can often find vector information at NCBI, either directly or in their list of vectors screened for contamination of new sequence at Vecscreen. VectorDB contains information about many common vectors, including yeast vectors. The EMBL Hamburg outstation maintains a large database of vectors. I.M.A.G.E. Consortium Vectors contains information about plasmids commonly used in EST collections such as those sold by OpenBioSystems and Invitrogen. For eukaryotic vectors (Fish, Xenopus) see Minnesota. The Forsburg Lab maintains a list of Fisson Yeast vectors. Promega maintains a list of their vectors. NEB maintains a list of common vectors. Epicentre also maintains its own list. Lucigen provides transcription-free vectors for cloning AT-rich and other difficult DNAs. Addgenes Vector DB contains most of the information from Stanfords VectorDB, plus more vector information they have curated from commercial websites and added through our plasmid curation efforts. (However, it seems to be rather ...
TY - JOUR. T1 - Induction of endogenous genes following infection of human endothelial cells with an E1- E4+ adenovirus gene transfer vector. AU - Ramalingam, Ramachandran. AU - Rafii, Shahin. AU - Worgall, Stefan. AU - Hackett, Neil R.. AU - Crystal, Ronald. PY - 1999/12/1. Y1 - 1999/12/1. N2 - Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1- E4+ Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding-protein ...
SB vs Viral Based Methods: "Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the hosts chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the ...
SB vs Viral Based Methods: "Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the hosts chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the ...
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγpos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF ...
Our viral vector production staff are committed to providing the researcher with high purity, high titer viral vectors at competitive prices. To ensure we the h
OriGene provides a variety of cloning vectors, including cDNA cloning vectors (tagged ORF clone vectors, non-tagged cDNA expression vectors), shRNA vectors, CRISPR vectors, miRNA expression vector and 3 UTR luciferase assay vector. These vectors have been used in almost 1,000 citations.
About the Journal Human Gene Therapy, the Official Journal of the European Society of Gene and Cell Therapy, British Society for Gene and Cell Therapy, French Society of Cell and Gene Therapy, German Society of Gene Therapy, and five other gene therapy societies, is an authoritative peer-reviewed journal published monthly in print and online. Led by Editor-in-Chief Terence R. Flotte, MD, Celia and Isaac Haidak Professor of Medical Education and Dean, Provost, and Executive Deputy Chancellor, University of Massachusetts Medical School, Human Gene Therapy presents reports on the transfer and expression of genes in mammals, including humans. Related topics include improvements in vector development, delivery systems, and animal models, particularly in the areas of cancer, heart disease, viral disease, genetic disease, and neurological disease, as well as ethical, legal, and regulatory issues related to the gene transfer in humans. Its companion journals, Human Gene Therapy Methods, published ...
Viral production. EIAV self-inactivating vector genomes were constructed from pONY8.0Z or pONY8.0G vectors described previously (10, 24). The cDNA coding for the reporter gene LacZ or the human SMN was cloned in the EIAV transfer vector. A EIAV vector genome has been generated that encodes the SMN gene with or without a HA tag. Viral vector stocks pseudotyped with rabies G glycoprotein were prepared using the HEK293T transient system as previously described (10, 25). The titers (approximately 4 × 109 T.U./ml) of concentrated EIAV-LacZ viral vectors were estimated by transduction of D17 cells. The titers (4 × 109 to 7 × 109 T.U./ml) of the EIAV-SMN or EIAV-EIAV-SMN-HA vectors were estimated using real-time quantitative RT-PCR by comparison to EIAV-LacZ vectors and were normalized for viral RNA (26, 27).. In vitro transduction. GM03813 fibroblasts from a type 1 SMA patient (Coreill Cell Repository) were transduced with either EIAV-SMN, EIAV-SMN-HA, or EIAV-LacZ vectors pseudotyped with the ...
The goal of the Program in Human Gene Therapy is to develop gene transfer technologies and use them for hepatic gene therapy for the treatment of genetic and acquired diseases. The general approach is to develop new vector systems and delivery methods, test them in the appropriate animal models, uncover the mechanisms involved in vector transduction, and use the most promising approaches in clinical trials. Specifically, we work on a variety of viral and non-viral vector systems. Our major disease models are hemophilia, hepatitis C and B viral infections, and diabetes. The second major focus includes the role that small RNAs play in mammalian gene regulation.. ...
The goal of the Program in Human Gene Therapy is to develop gene transfer technologies and use them for hepatic gene therapy for the treatment of genetic and acquired diseases. The general approach is to develop new vector systems and delivery methods, test them in the appropriate animal models, uncover the mechanisms involved in vector transduction, and use the most promising approaches in clinical trials. Specifically, we work on a variety of viral and non-viral vector systems. Our major disease models are hemophilia, hepatitis C and B viral infections, and diabetes. The second major focus includes the role that small RNAs play in mammalian gene regulation.. ...
riyer at rascal.med.harvard.edu wrote: : I recd. so many requests from people who did not get my earlier post that I : decided to save time by re-posting it. At the outset I would like to state : that I am only a satisfied user of the above vectors and am not associated : with the company that markets them. And now here comes a readable version (with ,80 chars/line) :-) : Hi netters, I would like to report the availability of a set of : reporter gene vectors that have some distinct advantages over those : currently in use for the purpose of analysis of eukaryotic promoters : & enhancers. The vectors , SV40-Syncat, Syncat I, Syncat II, : SV40-pFlash, pFlash I and pFlash II, are as their names suggest : CAT & Luciferase (Luc) reporter gene vectors. What distinguishes : them from other vectors in this category is that these vectors produce : near-zero background. This feature is due to the fact, that they use a : modified SV40-t-intron as the donor for the poly-adenylation signal. : The wild type ...
In article ,2gt1m4$o1r at hsdndev.harvard.edu, riyer at rascal.med.harvard.edu writes: ,I recd. so many requests from people who did not get my earlier post that I ,decided to save time by re-posting it. At the outset I would like to state ,that I am only a satisfied user of the above vectors and am not associated with the company that markets them. , , ,pow ,Hi netters, I would like to report the availability of a set of reporter gene vectors that have some distinct advantages over those currently in use for the purpose of analysis of eukaryotic promoters & enhancers. The vectors , SV40-Syncat, Syncat I, Syncat II, SV40-pFlash, pFlash I and pFlash II, are as their names suggest CAT & Luciferase (Luc) reporter gene vectors. What distinguishes them from other vectors in this category is that these vectors produce near-zero background. This feature , , ,to the fact, that they use a modified SV40-t-intron as the donor for the poly-adenylation signal. The wild type SV40-t-intron is the generic donor ...
Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV) to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired
You can often find vector information at NCBI, either directly or in their list of vectors screened for contamination of new sequence at Vecscreen. VectorDB contains information about many common vectors, including yeast vectors. EMBL maintains a large database of vectors. For eukaryotic vectors (Fish, Xenopus) see Minnesota. The Forsburg Lab maintains a list of Fisson Yeast vectors. Promega maintains a list of their vectors. NEB maintains a list of common vectors. Epicentre also maintains its own list. Addgenes Vector DB contains most of the information from Stanfords VectorDB, plus more vector information they have curated from commercial websites and added through our plasmid curation efforts. (However, it seems to be rather sparse when it comes to Escherichia coli vectors.) Note that Addgene is a a non-profit plasmid repository where scientists can archive and share their plasmids. They encourage and invite labs to deposit plasmids at Addgene. They help you with data submission and all ...
The Retro-X Universal Packaging System, featuring the GP2-293 packaging cell line, allows you to cater the tropism of the packaged virus to your target cell line. The envelope protein of your choice is cotransfected with your retroviral expression vector.
The Retro-X Universal Packaging System, featuring the GP2-293 packaging cell line, allows you to cater the tropism of the packaged virus to your target cell line. The envelope protein of your choice is cotransfected with your retroviral expression vector.
Understanding regulation of developmental events has increasingly required the use of tissue-specific expression of diverse factors affecting plant growth and environmental responses. To allow for cloning of presumptive promoters with tissue-specific activities, we created two plant expression vectors with multiple cloning sites upstream of a Gateway cassette for expression of either untagged or YFP-tagged genes of interest (Michniewicz et al., 2015) by modifying the pEarleyGate100 and pEarleyGate104 constructs (Earley et al., 2006). For fast and easy tissue-specific expression of desired genes, we further developed a set of Gateway-compatible tissue-specific gene expression vectors that allow for the expression of YFP-tagged or untagged proteins driven by various promoters (see table below). Additional vectors will be added to this table as they are created. These vectors provide an invaluable resource to the plant community, allowing for rapid generation of a variety of tissue-specific ...
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Since the development of vaccinia virus as a vaccine vector in 1984, the utility of numerous viruses in vaccination strategies has been explored. In recent years, key improvements to existing vectors such as those based on adenovirus have led to significant improvements in immunogenicity and efficacy. Furthermore, exciting new vectors that exploit viruses such as cytomegalovirus (CMV) and vesicular stomatitis virus (VSV) have emerged. Herein, we summarize these recent developments in viral vector technologies, focusing on novel vectors based on CMV, VSV, measles and modified adenovirus. We discuss the potential utility of these exciting approaches in eliciting protection against infectious diseases ...
The project work began with replacement of the original MCS for a completely new designed MCS containing the typical RFC10/25 pre- and suffixes. Furthermore we integrated a Strep-tag II coding gene sequence previous to the suffix. This facilitates purification and detection via Western Blot of expressed enzymes dramatically. Construction of the new MCS by four desoxyribooligonucleotides via oligonucleotide hybridization and ligation into the original pYES vector was restricted with the outermost restriction enzymes of the old MCS. Per side directed mutagenesis all forbidden restriction sides of enzymes used in RFC10/25 standard in the vector backbone were deleted. Different vector samples of this successive process are shown in the picture below. Their have been digested by NgoMIV, PstI and SpeI: The resulting fragments were decreased with each step leading to the pure vector backbone linearized by cutting in MCS. Hence our first original galactose inducible pGal1 promoter containing expression ...
Background: Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and posttranscriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter. Results: By exploiting a new method of producing the retroviral genome in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods. We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the ...
ASimilar packaging cells based on the use of T671 cells have been established by the same authors and have been named TeFLY. puro 2 × 105 TU/mL Ref. Rev (transduction by non-SIN MLV vectors) (CMV in the 5LTR tatindependent) SIN, eGFP VSV/G (transient transfection) VSV/G Non-SIN, eGFP MLV ampho, Non-SIN, eGFP GALV, and RD114 Env SIN, eGFP 293 3 × 105 TU/mL 146 293 2 × 105 TU/mL 147 HeLa, 293T HT1080 1 × 107 TU/mL 163 3 × 105 TU/mL 33 With the exception of one report that concerns the SIV LV genus (147), all other publications describe HIV-1-derived packaging cell lines. And Lynch, C. M. (1993) Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 217, 581-599. 15. , et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267. 20 Fluri and Fussenegger 16. Bukovsky, A. , Song, J. , and Naldini, L. (1999) Interaction of human immunodeficiency virus-derived vectors with wild-type virus in transduced cells. ...
Using the pYES vector from Invitrogen we first deleted five forbidden restriction sites in the vector back bone via side directed mutagenesis. Furthermore the original multiple cloning site was replaced for a multiple cloning site compatible to the RFC 10/25 cloning standards. To allow easy extraction and purification of proteins for in vitro applications the new multiple cloning site allows to express proteins with a Strep-tag II. Exclusion of the galactose inducible promoter provided a powerful basis vector for the integration of user-defined promoters. This way the pTUM100 vector gives a valuable contribution to our and to further protein expression and promoter characterization experiments in the yeast Saccharomyces cerevisiae. Moreover we used the pTUM100 to integrate the three constitutive promoters Tef1, Tef2 and ADH which come all with different promoter intensities. ...