G pseudotyping and transduction efficiency of lentiviral vectors. Production, concentration and titration of pseudotyped hiv. Efficient largescale production and concentration of hiv 1based lentiviral vectors for use in vivo jason e. Production and titration of lentiviral vectors barde. Simplified production of lentiviral vectors using hyperflasks. Nature publishing group, london, uk pubmed reiser j, harmison g, kluepfelstahl s, brady ro, karlsson s, schubert m 1996 transduction of nondividing cells using pseudotyped defective hightiter hiv type 1 particles. Since their development nearly fifteen years ago, vsvg pseudotyped selfinactivating sin lentiviral vectors lvs have become an indispensible part of the experimental biologists toolbox and have met with success in clinical gene therapy trials naldini et al. Production and concentration of pseudotyped hiv1based vectors article pdf available in gene therapy 711. The potential for generation of replicationcompetent lentivirus is addressed by the design of the vectors and by safe laboratory practice. A variety of protocols are available for concentration of lvs.
Lentiviral vector production systems are based on a split system, where the natural viral genome has been split into individual helper plasmid constructs. Reiser j 2000 production and concentration of pseudotyped hiv1based gene transfer vectors, vol 7. We investigated the production efficiency and the gene transfer capacity in the central nervous system of hiv 1based vectors pseudotyped with either the g protein of the mokola lyssaviruses mkg, a neurotropic virus causing rabies disease, or the vesiculostomatitis g protein vsvg. These data indicate that the minimum required concentration of virion.
Simplified production and concentration of hiv1based. The particular characteristics of lvs allied to their marked development during the last years have triggered the attention of different fields, consequently a vast range of applications for these vectors. Production and concentration of pseudotyped hiv 1based gene transfer vectors. For lentivirus vectors encoding fluorescent proteins under the control of promoters functional in these cells, titration can be performed using the limiting dilution method or a flow cytometry. An optimized method for hightiter lentivirus preparations without ultracentrifugation. Production and purification of lentiviral vectors nature.
Thus, like lentiviral vectors pseudotyped with vsvg, hiv1based vectors pseudotyped with the mlv 4070a amphotropic env and the rabies g glycoprotein resist inactivation during concentration. Production, purification and titration of a lentivirus. Pdf production, concentration and titration of pseudotyped hiv1. The production of a third generation hiv 1based lentiviral vector, expressing an enhanced green. The advantages of using hiv 1based vectors over other vector types for a ex vivo autologous cd4 tcellbased hiv gene therapy are. Based on these results we have proceeded to examine the tropism of chikvg pseudotyped lentiviral vectors more extensively. Selective transduction of astrocytic and neuronal cns. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells.
This is an hek 293tderived cell line optimized for lentix virus production. Mucosal and submucosal edema was found in the gastrointestinal tract of 14 treated and 6 control mice, and congestion was observed in the livers of both control and treated mice. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. Viral gene delivery is hailed as a great milestone in genebased therapeutic approaches. The human immunodeficiency virusderived lentiviral vectors lvs are advantageous in infecting both dividing and nondividing cells leading to continuous expression of transgenes. Production, concentration and titration of pseudotyped hiv 1based lentiviral vectors 19 march 2009 nature protocols, vol. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines. The third generation lentiviral vector backbone contains an egfp reporter anderson et al. Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins gps derived from. The superiority of sucrose cushion centrifugation to. Thus, like lentiviral vectors pseudotyped with vsvg, hiv 1based vectors pseudotyped with the mlv 4070a amphotropic env and the rabies g glycoprotein resist inactivation during concentration. Pdf production and concentration of pseudotyped hiv1. Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells.
Protocolqualitative detection of norovirus and hepatitis a virus in soft fruit. Lentiviral vectors have emerged as powerful and versatile vectors for ex vivo and in vivo gene transfer into dividing and nondividing cells. Filovirus pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo. Production and concentration of pseudotyped hiv1 based. Cellculture assays reveal the importance of retroviral vector design for insertional genotoxicity. Production and titration of lentiviral vectors current. Lentiviral vector production, titration, and transduction.
How can i do the titration of the lentivirus with qpcr. An optimized method for hightiter lentivirus preparations. Production and concentration of pseudotyped hiv1based gene transfer vectors. Production, concentration and titration of pseudotyped hiv1based lentiviral vectors. Good recov eries have been reported based on this procedure. Production, purification and titration of a lentivirusbased vector for. In terms of vector design, 2nd and 3rd generation lentiviral systems provided by addgene separate transfer, envelope, and packaging components of the virus onto different vectors. With a view toward improving hightiter lv vector production for preclinical studies in animals, we tested the usefulness of hyperflask vessels that have a total growth area of 1720 cm 2, corresponding to ten standard t175 flasks. Assessment of selected media supplements to improve fhn. Cell lines for lentivirus packaging and titration lentix 293t cell line cat. Production, concentration and titration of pseudotyped hiv 1based lentiviral vectors article pdf available in nature protocol 44. There was no loss in titer with any of the pseudotypes tested. Tropism of the chikvg pseudotyped lv was assessed via comparison of its biological titre obtained on different cells lines fig.
Lentiviral vectors lv have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. They were incorporated into vector particles and mature. Production and concentration of pseudotyped hiv1based gene. Production, concentration and titration of pseudotyped hiv 1based lentiviral vectors. Lentiviral vectors for hiv disease prevention and treatment. Pseudotyping of lentiviral vector with novel vesiculovirus. They can transduce a wide range of cell types and integrate into the. Production, concentration and titration of pseudotyped hiv 1based lentiviral vectors skip to main content thank you for visiting. Many such viruses have been the basis of research using viruses in gene therapy, but the lentivirus is unique in its. The heterologous envelope gene containing plasmids vsvg, cnvg and prvg were used to produce pseudotyped lentiviral vectors. Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentivirus lentivirus are a family of viruses that are responsible for notable diseases like hiv, which infect by inserting dna into their host cells genome. Production and concentration of pseudotyped hiv 1based vectors article pdf available in gene therapy 711. Production and titration of lentiviral vectors salmon. Thus, like lentiviral vectors pseudotyped with vsvg, hiv1based vectors.
Production, concentration and titration of pseudotyped hiv1based lentivirus vectors. Efficient largescale production and concentration of hiv. Production, concentration and titration of pseudotyped hiv 1based lentivirus vectors. Production, concentration and titration of pseudotyped hiv1based lentiviral vectors article pdf available in nature protocol 44. Simplified production and concentration of lentiviral vectors to. These production concentration methods result in hightiter vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugationbased methods. In addition to the standard 293t cells, the neuronal mouse neuro2a and human shsy5y cell lines as well as normal human astrocytes nha.
Biodistribution and toxicity studies of vsvgpseudotyped. The vector production and titration protocol described here can be completed within 8 d. Production and concentration of pseudotyped hiv1based. We primarily generated our internal ribosome entry site ires.
Methods based on anion exchange column chromatography of lv vectors pseudotyped with vsvg 17, 18 or the baculovirus gp64 glycoprotein. Production, purification and titration of a lentivirus based vector for gene delivery purposes. Vector concentration and administration to assess the potency of the firstgeneration lentiviral vector hr cmvgfp, vector stocks from several batches of inductive production and concentration were thawed at 30 c and pooled. Production, concentration and titration of pseudotyped hiv1based. Reiser j 2000 production and concentration of pseudotyped hiv 1based gene transfer vectors, vol 7. Hyperflasks consist of ten interconnected growth surfaces each containing a membrane pretreated to. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replicationcapable virus by adventitious recombination of the lentiviral genome. Nature publishing group, london, uk reiser j, harmison g, kluepfelstahl s, brady ro, karlsson s, schubert m 1996 transduction of nondividing cells using pseudotyped defective hightiter hiv type 1 particles. Lentivirus production with the lentix packaging single shots and lentix 293t cells. Production, concentration and titration of pseudotyped hiv1based lentiviral vectors skip to main content thank you for visiting. Transduction was evaluated with both human 293 cells and mouse nih 3t3 cells by facs analysis for gfp expression table 1. Simplified production and concentration of hiv1based lentiviral.
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