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
A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.. ...
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, ...
We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis-acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.
Of the 127 allogeneic trials analysed by Evolution, over two-thirds (69.3%) utilise an adeno-associated viral vector, followed by adenoviruses (10.2%), plasmids (9.4%) and ADV/HSV-tk (3.9%). Lentiviruses, retroviruses and herpes simplex viruses were each used in three clinical trials for allogeneic cell/gene therapies, representing 2.4% each.. With regards to the 82 autologous cell/gene therapies currently undergoing clinical trials, 59.8% utilise a lentivirus and 30.5% utilise a retrovirus. In total, 74 of the 82 active trials analysed by Evolution utilise either a lentiviral or retroviral vector. Five (6.1%) active trials utilise an adenoviral vector and two (2.4%) utilise an HSV vector. In stark contrast to allogeneic approaches, adeno-associated vectors (AAV) and plasmids are not utilised in any of the ongoing autologous trials.. Lentiviruses are a subclass of retroviruses and are comprised of an envelope, a capsid and an RNA genome. In the transduced cell, the RNA genome is ...
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.
Nanotechnology-based non-viral vectors have emerged as promising alternatives to viruses to carry genetic material into target cells due to their ability to overcome many limitations of viral vectors. Although very efficient, viruses present low carrying capacity, expensive and complex production and, most importantly, safety issues related to immunogenic responses and even oncogenesis when randomly integrated in the host genome. In this regard, non-viral vectors present lower immunogenicity, higher nucleic acid packing capacity and ease of fabrication compared to their viral counterparts. In addition, continuous advances in the fields of material science and nano-engineering as well as the diversity of available nanosized material allows the design of multifunctional vectors specifically tailored for different applications. Although most conventional gene therapy strategies based on gene addition use viral vectors, nowadays non-viral vectors are predominant in new investigations based on gene ...
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 ...
However, viral vector-based vaccines that incorporate viruses other than SARS-CoV-2 are in advanced-phase studies, including adenovirus (AdV) vector-based vaccines that have been licensed in Europe. These vaccines consist of intact virions that are engineered to include the gene encoding the SARS-CoV-2 spike protein, a technique that leverages the viral vectors ability to efficiently infect cells and enhances spike gene delivery. Vaccines that use viral vectors contain either replication-deficient or replication-competent viruses (Table 1). The majority of viral-vectored vaccines in the most advanced phases of development have been rendered replication-deficient through deletion of genes essential for replication.8 By limiting vector replication, the potential for vaccine-associated AdV disease is greatly diminished.. There are, however, theoretical mechanisms by which replication-deficient viral vector-based vaccines could become replication competent and cause disease, especially in ...
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
We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this tech …
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 ...
TY - JOUR. T1 - In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector. AU - Kubo, Shuji. AU - Kataoka, Miho. AU - Tateno, Chise. AU - Yoshizato, Katsutoshi. AU - Kawasaki, Yoshiko. AU - Kimura, Takahiro. AU - Faure-Kumar, Emmanuelle. AU - Palmer, Donna J.. AU - Ng, Philip. AU - Okamura, Haruki. AU - Kasahara, Noriyuki. PY - 2010/1/1. Y1 - 2010/1/1. N2 - We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved ...
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 - Targeting tumor neoangiogenesis via targeted adenoviral vector to achieve effective cancer gene therapy for disseminated neoplastic disease. AU - Lee, Myungeun. AU - Lu, Zhi Hong. AU - Li, Jie. AU - Kashentseva, Elena A.. AU - Dmitriev, Igor P.. AU - Mendonca, Samir A.. AU - Curiel, David T.. PY - 2020/3. Y1 - 2020/3. N2 - The application of cancer gene therapy has heretofore been restricted to local, or locoregional, neoplastic disease contexts. This is owing to the lack of gene transfer vectors, which embody the requisite target cell selectivity in vivo required for metastatic disease applications. To this end, we have explored novel vector engineering paradigms to adapt adenovirus for this purpose. Our novel strategy exploits three distinct targeting modalities that operate in functional synergy. Transcriptional targeting is achieved via the hROBO4 promoter, which restricts transgene expression to proliferative vascular endothelium. Viral binding is modified by incorporation ...
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 ...
TY - JOUR. T1 - An effective gene-knockdown using multiple shRNA-expressing adenovirus vectors. AU - Motegi, Yukari. AU - Katayama, Kazufumi. AU - Sakurai, Fuminori. AU - Kato, Takuya. AU - Yamaguchi, Tomoko. AU - Matsui, Hayato. AU - Takahashi, Masahide. AU - Kawabata, Kenji. AU - Mizuguchi, Hiroyuki. PY - 2011/7/30. Y1 - 2011/7/30. N2 - Viral vectors expressing short hairpin RNA (shRNA) are attractive for efficient and tissue-specific RNA interference (RNAi) delivery. We and others previously reported that recombinant adenovirus (Ad) vector-mediated RNAi has great potential for a variety of applications in molecular biology studies and gene therapy. In the present study, we have developed an efficient Ad vector-mediated RNAi system, in which an Ad vector carries four shRNA-expression cassettes (Ad-multi-shRNA vector), a simple and effective strategy for enhancing the RNAi response per Ad vector particle. The data demonstrated that the Ad-multi-shRNA vectors showed an enhanced RNAi effect ...
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 ...
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Context General information Compatible Gateway Destination vectors Available clones Download the hORFeome V8.1 list Compatible Gateway Destination vectors BCCM/genecorner also offers a range of compatible Gateway destination vectors both with and without C- or N-terminal markers, as well as some multisite Gateway destination vectors. This way, you can create the expression
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.. ...
A new dual-vector approach to enhance recombinant adeno-associated virus-mediated gene expression through intermolecular cis activation
Adeno-associated virus is being developed as a human gene therapy vector. Recombinant AAV vectors are constructed by replacing the wild type viral genome with a transgene of interest. This recombinant genome is then packaged into infectious virions. Virion production requires a vector plasmid containing the transgene of interest, wtAAV rep and cap genes, and helper virus functions. Standard protocols provide rep and cap either through stable rep/cap cell lines, or through co-transfection of a rep/cap plasmid. Helper virus functions are provided by adenovirus-5 or herpes simplex virus-1. Viral infection results in loss of some recombinant virions due to cell death as well as potential contamination of the final stock. To overcome these problems, several helper-virus-free protocols have been developed using plasmids containing essential Ad-5 genes. Standard purification protocols involve CsCl gradient centrifugation followed by dialysis.The centrifugation takes several days, and product is lost in ...
The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was ...
Combined expression of costimulatory factors and proinflammatory cytokines stimulate effective immune-mediated tumor rejection in a variety of murine tumor models. Specifically, syngeneic tumor cells genetically modified to express B7.1 (CD80) have been shown to induce rejection of previously established murine solid tumors, and transduction with IL-2 can further increase survival. However, poor rates of gene transfer and inefficient expression of multiple transgenes encoded by single vectors have hampered the development of such autologous tumor cell vaccines for clinical trials in acute myeloid leukemia (AML) patients. Here we describe the development of a self-inactivating lentiviral vector encoding B7.1 and IL-2 as a single fusion protein postsynthetically cleaved to generate biologically active membrane-anchored B7.1 and secreted IL-2. This enables the efficient transduction of both established and primary AML blasts, resulting in expression of the transgenes in up to 98% of the cells following a
In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors Academic Article ...
Non-viral gene delivery vectors were developed for efficient gene transfer to hard to transfect mouse mammary epithelial cells. broad spectrum of human being diseases including malignancy, cardiac disorders and neurodegenerative diseases result from a genetic defect. Gene therapy gives an attractive option to treat these genetic diseases [1]. The introduction of an exogenous restorative gene into unhealthy cells offers the potential to override or change the malfunctioning gene. Although a large quantity of genetic focuses on possess been recognized, the medical success of gene therapy methods offers been limited by the lack of availability of safe and effective gene delivery vectors 3963-95-9 IC50 [2]. Two major attempts possess emerged in the gene delivery community, one focused on developing viral vectors and the additional on non-viral strategies. Viral vectors have developed to transduce many types of mammalian cells with high effectiveness, but there are limitations to cell-specificity, ...
A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins. ...
TY - JOUR. T1 - Inhibition of repair of radiation-induced DNA damage enhances gene expression from replication-defective adenoviral vectors. AU - Hingorani, Mohan. AU - White, Christine L.. AU - Merron, Andrew. AU - Peerlinck, Inge. AU - Gore, Martin E.. AU - Slade, Andrew. AU - Scott, Simon D.. AU - Nutting, Christopher M.. AU - Pandha, Hardev S.. AU - Melcher, Alan A.. AU - Vile, Richard Geoffrey. AU - Vassaux, Georges. AU - Harrington, Kevin J.. PY - 2008/12/1. Y1 - 2008/12/1. N2 - Radiation has been shown to up-regulate gene expression from adenoviral vectors in previous studies. In the current study, we show that radiation-induced dsDNA breaks and subsequent signaling through the mitogen-activated protein kinase (MAPK) pathway are responsible, at least in part, for this enhancement of transgene expression both in vitro and in vivo. Inhibitors of ataxia-telangiectasia-mutated, poly(ADP-ribose) polymerase-mutated, and DNA-dependent protein kinase (DNA-PK)-mediated DNA repair were shown to ...
A recombinant adeno-associated virus serotype 2 (rAAV2) vector has been altered to carry the human MERTK (hMERTK) gene. This vector has been shown to restore vision in animal models that resemble human MERTK-associated Retinitis Pigmentosa (RP), an incurable retinal degeneration that causes severe vision loss. The proposed study is an open label, Phase I clinical trial of subretinal rAAV2-VMD2-hMERTK administration to individuals with MERTK-associated retinal disease. This trial will lead to a greater understanding of the safety and thereby potential value of gene transfer in MERTK-associated retinal disease and will have implications for other forms of retinal degenerative disease amenable to this type of intervention ...
A recombinant adeno-associated virus serotype 2 (rAAV2) vector has been altered to carry the human MERTK (hMERTK) gene. This vector has been shown to restore vision in animal models that resemble human MERTK-associated Retinitis Pigmentosa (RP), an incurable retinal degeneration that causes severe vision loss. The proposed study is an open label, Phase I clinical trial of subretinal rAAV2-VMD2-hMERTK administration to individuals with MERTK-associated retinal disease. This trial will lead to a greater understanding of the safety and thereby potential value of gene transfer in MERTK-associated retinal disease and will have implications for other forms of retinal degenerative disease amenable to this type of intervention ...
The effect of chemotherapy drug Mitomycin C (MMC) in combination with recombinant adeno-associated virus II (rAAV2) in cancer therapy was investigated, and the mechanism of MMC affecting rAAV2s bioactivity was also studied. The combination effect was evaluated by the level of GFP and TNF expression in a human glioma cell line, and the mechanism of MMC effects on rAAV mediated gene expression was investigated by AAV transduction related signal molecules. C57 and BALB/c nude mice were injected with rAAV-EGFP or rAAV-TNF alone, or mixed with MMC, to evaluate the effect of MMC on AAV-mediated gene expression and tumor suppression. MMC was shown to improve the infection activity of rAAV2 both in vitro and in vivo. Enhancement was found to be independent of initial rAAV2 receptor binding stage or subsequent second-strand synthesis of target DNA, but was related to cell cycle retardation followed by blocked genome degradation. In vivo injection of MMC combined with rAAV2 into the tumors of the animals
294966285 - EP 0996737 A1 2000-05-03 - SEMLIKI FOREST VIRUS VECTORS FOR GENE TRANSFER INTO NON-ENDOTHELIAL CARDIOVASCULAR CELLS - [origin: WO9909192A1] The invention provides methods of transducing non-endothelial cardiovascular cells with a recombinant Togavirus vector, vector kits, and vectors.[origin: WO9909192A1] The invention provides methods of transducing non-endothelial cardiovascular cells with a recombinant Togavirus vector, vector kits, and vectors.
Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. MLV vectors, whereby the last two are transcriptionally restricted to liver- and -catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from contaminated cells was significantly decreased. Fusion from the heterologous promoters missing the TATA container towards the MLV TATA container, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting negligible or low spread in nonpermissive cells. The genomic balance from the vectors was been shown to be much like that of an identical vector filled ...
Because the features and kinetics of adeno−associated virus(AAV)−mediated gene transfer to endothelial cells(EC)are yet to be ultimately determined, we tested variables pertinent to the efficiency of AAV−mediated gene transfer to bovine aortic endothelial cells(BAEC).The variables with AAV vectors were compared with the better characterized adenovirus(Ad)vectors.There is a dose−response relationship between multiplicity of infection(moi)of AAV or Ad vectors and transduction efficiency in BAEC.The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured BAEC.AAV and Ad vectors showed an incubation−time−dependent increase in transduction efficiency of LacZ gene to the BAEC up to 12 h of vector exposure.Although the similar kinetics of transduction efficiency of LacZ gene to BAEC was found in both vectors, the duration of gene expression was longer in AAV vector than that in Ad vectors in vitro.These results indicate that AAV−vector is efficient for gene ...
Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was ...
TY - JOUR. T1 - G2 Cell Cycle Arrest and Cyclophilin A in Lentiviral Gene Transfer. AU - Zhang, Shangming. AU - Joseph, Guiandre. AU - Pollok, Karen. AU - Berthoux, Lionel. AU - Sastry, Lakshmi. AU - Luban, Jeremy. AU - Cornetta, Kenneth. PY - 2006/10. Y1 - 2006/10. N2 - Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34+ peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, ...
Recombinant vectors based on adeno-associated virus (rAAV) are promising tools to specifically alter complex genomes through homologous recombination (HR)-based gene targeting. In a therapeutic setting, an AAV donor vector will recombine with a mutant target locus in order to correct the mutation di …
The airway is an important target for gene transfer to treat cystic fibrosis and other diseases that affect the lung. We previously found that marker gene expression did not persist in the bronchial epithelium following adeno-associated virus (AAV) vector administration to the rabbit lung. In an attempt to promote continued expression, we tested repeat vector administration, but no additional transduction was observed, and the block to transduction correlated with the appearance of neutralizing antibodies to the viral capsid. Here we show that mice exhibit a similar response but that treatment with anti-CD40 ligand antibody (MR1) and a soluble CTLA4-immunoglobulin fusion protein (CTLA4Ig) at the time of primary AAV vector exposure allowed successful repeat transduction and prevented production of neutralizing antibodies. We also tested the possibility that an immune response caused the loss of marker-positive cells in the epithelial population in rabbits by evaluating AAV vector expression in ...
AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. Recombinant AAV vectors delivering RPE65 gene have been demonstrated...
AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. Recombinant AAV vectors delivering RPE65 gene have been demonstrated...
Gene transfer to the gastrointestinal (GI) mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD), GI infections, and cancer. We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G)-pseudotyped lentiviral vectors (LV) for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal) or firefly-luciferase (LV-fLuc) reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression
Recombinant Adeno-associated virus 2 ATCC ® VR-1616™ Designation: Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2 RSS) Application:
Recombinant Adeno-associated virus 2 ATCC ® VR-1616™ Designation: Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2 RSS) Application:
This study evaluated six adeno-associated viral (AAV) vectors expressing green fluorescent protein (GFP) from the liver-specific thyroid hormone-binding globulin (TBG) promoter made with novel capsids in canine liver-directed gene transfer. Studies i
Background: Liver-derived Factor VIII (FVIII) expression has been described in the canine hemophilia A model for more than 10 years following adeno-associated viral (AAV) gene therapy. Improved understanding of AAV integration may provide insights into the long-term safety of AAV based gene therapy.. Aims: To evaluate frequency and locations of AAV-FVIII integration sites in a canine hemophilia model.. Methods: Eight severe hemophilia A dogs received a portal vein infusion of canine-FVIII-AAV vector (AAV-BDD-cFVIII). Genomic DNA was isolated from post-mortem liver samples. AAV-BDD-cFVIII vector genomes (vg/ng) were quantified by qPCR. Vector integration sites (IS) were analysed by linear amplification-mediated (LAM)-PCR and target-enrichment sequencing (TES) coupled to Illumina MiSeq sequencing and customised bioinformatical analysis.. Results: AAV-BDD-cFVIII DNA was detected in the liver of all dogs, 10.8 years after a single vector infusion (Table 1). LAM-PCR and TES analyses demonstrated ...
Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to
The snoMEN (snoRNA Modulator of gene Appearance) vector technology was developed from a human being package C/D snoRNA, HBII-180C, which contains an internal series that may end up being manipulated to help to make it supporting to RNA focuses on, allowing knock-down of targeted genetics. the same mRNA, we record >3-collapse boost in knock-down effectiveness when likened with the unique HBII-180C centered snoMEN. The multiplex 47snoMEM vector allowed the building of human being proteins replacement unit cell lines with improved effectiveness, including the institution of book GFPCHIF-1 alternative cells. Quantitative mass spectrometry evaluation verified the improved effectiveness and specificity of proteins replacement unit using the Solcitinib supplier 47snoMEN-PR vectors. The 47snoMEN vectors increase the potential applications for snoMEN technology in gene appearance research, focus on approval and gene therapy. Intro Little nucleolar RNAs (snoRNAs) are a course of conserved RNAs 1st ...
Custom RNAi Services GeneCust Europe. of shRNA expression plasmids,. shRNA expression vectors system characteristics 1.The expression of short. the duplication of the shRNA expression cassette resulted in. extracted with the SV total RNA isolation system (Promega.pHUSH: a single vector system for conditional gene expression pHUSH:. Reversible gene knockdown in mice using a tight, inducible shRNA expression system.Altered expression of triadin 95 causes parallel changes. this system adenoviral transfection was used to modify. endogenous Trisk 95 expression. The shRNA was.HeLa cells containing a stably integrated tetracycline-inducible luciferase expression cassette were treated with. The Tet-Express Inducible Expression System.Applications. The following. Modulation of Circadian Gene Expression and Metabolic Compensation by. Novel high throughput pooled shRNA screening identifies ...
Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Consequently, gene transfer studies in non-\human being primates may well forecast the effectiveness of gene transfer in humans. Indeed, gene transfer studies using a fresh type of vector have been carried out in rhesus macaques.12,13 The effects from these studies provided the basis for recent hemophilia B gene therapy clinical tests employing an AAV8 vector.13,14,15,16 Gene transfer in mice using AAV vectors results in excellent transduction efficiency. That is so for AAV8 vector-mediated gene transfer in the mouse liver especially;12,13,14,17 however, the efficiency of AAV8 vectors is modest in macaques.13 Theres also difficulties connected Mouse monoclonal to STYK1 with gene appearance when working with AAV8 vectors in non-human primates. Growing proof suggests that the current presence of neutralizing antibodies (NAbs) against AAV8, ...
Cornerstone for an efficient cardiac gene therapy is the need for a vector system which enables selective and long-term expression of the gene of interest. In rodent animal models pseudotyped adeno-associated viral vectors like AAV2/6 have been shown to be superior in the transduction of cardiomyocytes compared to other AAV serotypes. However, since significant species dependent differences in transduction characteristics exist, and large animal models are of imminent need for pre-clinical evaluations, we compared the gene transfer efficiency of an AAV2/6 and a heparin-binding site deleted AAV2 (AAV2/dHep) in a porcine model.. Methods: Viral vectors were produced using a pseudotyping strategy. All vectors did express the reporter gene luciferase under control of a cardiac specific promoter (MLC2v). Vectors were delivered via percutaneous pressure-regulated retroinfusion into the coronary vein (3.5 x 10e10 genome copies/animal; n = 5 animals/group). Expression levels were evaluated 4 weeks after ...
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RNA interference (RNAi) is a highly conserved post-transcriptional gene silencing process triggered by double-stranded RNA (dsRNA) in eukaryotic cells. Elucidation of the RNAi regulatory pathway and its components has led to the identification of endogenous dsRNA molecules, termed microRNAs (miRNAs), which are transcribed as a single hairpin molecule prior to their maturation into a cytoplasmic dsRNA. The efficient gene silencing achieved by these short hairpin RNA (shRNA) molecules and the cumulative understanding of the RNAi pathway has prompted the development of hairpin expression vectors capable of mediating stable gene silencing in vitro and in vivo. The aim of this thesis is to evaluate the efficacy of viral vector-mediated RNAi in the retina using recombinant adeno-associated viruses (AAV) and lentiviruses that contain silencing hairpin cassettes to target four genes in murine photoreceptors and the retinal pigment epithelium (RPE). A detailed assessment of the utility and extend of RNAi ...
Staining for LacZ 5 days after intraocular injection of AdLacZ.10 in adult wild-type mice and at various times after injection in transgenic mice with increased expression of PDGF in the retina. Mice were given an intravitreous injection of 5 × 108 (A-E, J-P) or a subretinal injection of 107 (F-I) AdLacZ.10 particles. After 5 days, or as otherwise indicated, ocular sections and retinal and choroidal wholemounts were stained for LacZ. (A) Albino BALB/c mice showed staining in the corneal endothelium, the trabecular meshwork, the iris pigmented epithelium, and the ciliary body (bar, 200 μm). (B) Flatmounts of iris from albino BALB/c mice showed diffuse dark staining throughout the entire iris. (C.) Retinal wholemounts from adult C57BL/6 mice showed scattered focal staining throughout the retina, with more intense staining at the optic nerve. (D, E) Ocular sections from adult C57BL/6 mice showed prominent staining in and around the optic nerve and focal staining of cells in the inner nuclear ...
TY - JOUR. T1 - Radioiodinated Capsids Facilitate in Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors. AU - Kothari, P.. AU - De, B. P.. AU - He, B.. AU - Chen, A.. AU - Chiuchiolo, M. J.. AU - Kim, D.. AU - Nikolopoulou, A.. AU - Amor-Coarasa, A.. AU - Dyke, J. P.. AU - Voss, H. U.. AU - Kaminsky, S. M.. AU - Foley, C. P.. AU - Vallabhajosula, S.. AU - Hu, B.. AU - DiMagno, S. G.. AU - Sondhi, D.. AU - Crystal, R. G.. AU - Babich, J. W.. AU - Ballon, D.. N1 - Publisher Copyright: © The Author(s) 2017. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.. PY - 2017/1/6. Y1 - 2017/1/6. N2 - Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here ...
Lentiviral vector systems with EF-1 alpha promoters allow you to achieve robust, constitutive, long-term expression of your gene of interest in cell types in which CMV promoters are often silenced, such as hematopoietic and stem cells.
Lentiviral vector systems with EF-1 alpha promoters allow you to achieve robust, constitutive, long-term expression of your gene of interest in cell types in which CMV promoters are often silenced, such as hematopoietic and stem cells.