Invitrogen lipofectamine 2000 reagent, and pei in a 6well culture plate with hek 293tft cells. Detailed protocol for lentiviral production using lipofectamine 3000 reagent. Lentivirus production with the lentix packaging single shots and lentix 293t cells. Allows production of a lentiviral construct that facilitates expression of a gene of interest under the control of a promoter of choice. Production of lentiviral vectors for transducing cells from.
Currently, most protocols for generating hightiter lentivirus require. An optimized method for hightiter lentivirus preparations. Ilv is the traditional lentivirus which can integrate the viral cdna into its target cell genome to form proviral dna. Review and cite lentivirus production protocol, troubleshooting and other methodology information contact experts in lentivirus production to get answers. A workflow is provided in figure 2, followed by a more detailed protocol. Advantages of lentiviral production using lipofectamine 3000 reagent.
The lentivirus produced in this protocol can be used to create transgenic quail, as described in injection. Lentiviral expression system, continued the nativepure lentiviral expression system is derived from the virapower lentiviral expression system, which facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and nondividing mammalian cells using a replicationincompetent lentivirus. It has been shown that a variety of production systems ef. The vsvg envelope protein is commonly used in lentiviral particle production because it confers broad tropism over a range of species and cell types. In general, the dna yield is around 3050 g for each miniprep. Lentiviral vectors lv have been used by many investigators to modify cells in vitro and in vivo, because they can integrate a transgene or shrna into the genome of most cell types 1. This protocol describes how to produce lentiviruses using hek293 cells, and is. Two new protocols to enhance the production and isolation of. This protocol can be used to produce lentivirus from a lentiviral vector transfected into lentix 293t cells using a polyethyenimine pei transfection protocol.
Specifically, id like to know what the general ratios you use for packaging, envelope, and transfer plasmids are. Two hrs prior to transfection, medium was replaced with rpmi 1640 supplemented with 5% fcs. Add the transfection mix dropwise being careful not to dislodge the cells. User manual please refer to storage conditions in manual a limiteduse label license covers this. Virabind lentivirus concentration and purification kit does not involve ultracentrifugation. The following protocol describes the general procedure for generation of pseudoviral packaged lentiviral constructs using thermofishers invitrogen lipofectamine and plus reagent see additional materials for production of lentivirus.
We have found that antibiotics pepstrep do not affect the ability of lipofectamine reagents to transfect cells. Protocol 2 producing lentivirus in hek293t cells using a 2. Lentiviral based gene transduction and promoter studies in human hematopoietic stem cells hhscs. The lentiviral plasmid contains the dna the virus will insert into the genome of every cell it infects, while the packaging vectors contain genes for all the other proteins required to make a lentivirus. Improve lentiviral production using lipofectamine 3000 reagent. Welgens proprietary lentiviral system is highly efficient to produce stable cell lines with any gene and any host cell, including mammalian primary cell. Comparison of genjet plus in vitro dna transfection reagent with leading products including lipofectamine 2000 l2k and lipofectamine ltx ltx on lentivirus production on 293t cells. Transfect hek293t t75, 90% confluent cells with 21ug of total dna 3 dna constructs using lipofectamine ltx 105ul. Mix each tube well by vortexing or flicking the tube. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering. As with standard plasmid vectors, it is possible to introduce lentiviral constructs in plasmid form into the cells with lowtomedium. Please refer to the user manual when the lentivirus expression systems from invitrogen or system biosciences are used. Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and nondividing cells. Lentix lentiviral expression system user manual pt551 062619 lentix lentiviral expression systems user manual.
Invitrogen lipofectamine 3000 transfection reagent can help improve lentivirus production. Titers of 10 8 can be achieved with an optimized protocol. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i. Lentiviral vector production thermo fisher scientific ca. Transitlenti transfection reagent high titer lentivirus. For instructions to thaw, culture, and maintain the 293ft cell line, see the 293ft cell line manual. Improve lentiviral production using lipofectamine 3000. The average lentiviral titers in our preparations are around 56x 10 5 x 10. Lentiviral vector production for cell and gene therapy applications. Is it ok to use antibiotics in the transfection media when. Do you have a toxicity problem with lipofectamine 2000. The goodell laboratory author karen lin july 30, 2005 title production of mscv retrovirus introduction the objective of this protocol is to transfect 293t cells and produce high titer of mscv viruses in a 6well plate. The proviral dna can replicate together with cell genome during cell dividing and exist in each new cell. Lentivirus retrovirus based stable cell line development services highly efficient with any gene and any host cell, including mammalian primary cells, for cellbased assays testing functions of chemicals, proteins, antibodies, cart cells, and carnk cells.
Whether youre a novice or an expert, you can rely on these benefits. The virapower and virapower ii lentiviral expression kits include the virapower lentiviral support kit plus an expression vector and the. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. In a sidebyside comparison between lipofectamine 3000 reagent, lipofectamine 2000 reagent and pei, lipofectamine 3000 reagentmediated lentiviral production was 510 times greater than that obtained from the other transfection reagents figure 5.
If lot screening is not an option, then hyclone sourced fbs is typically a safe bet. Lentivirus was collected 48 hours posttransfection and concentrated by prolonged centrifugation at 9,000 x g. Apr, 2016 lentiviral vectors lv have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. The blockit inducible h1 lentiviral rnai system combines blockit rnai and virapower trex lentiviral technologies to facilitate creation of a replicationincompetent lentivirus that delivers an inducible short hairpin rna shrna of interest to dividing or nondividing mammalian cells for rna interference rnai analysis. None of the protocol i see reference a specific product. The following morning, carefully aspirate the media. For more information about the 293ft cell line, refer to the 293ft cell line manual. Im relatively new to viral transduction, and im looking to see if anyone has experience with lipofectamine 2000 based lentivirus production in 293t cells who can assist me. Production of lentiviral stocks requires packaging of the lentiviral genomic rna. Produce hightiter lentivirus with this hivbased lentivector packaging kit. Lipofectamine 2000 please follow suggested guidelines for 150mm plates. This feature makes ilv an ideal tool for stable cell line generation to allow longterm gene expression. Hello everybody i am currently trying to establish lentivirus production in hek293t cell line.
Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. Check the cells 12 days after transfection to determine what ratio gives the highest percentage of gfp positive cells. The transfection reagent lipofectamine 2000 lf2000, invitrogen is. The packaging cells produce infectious particles, whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the target cells.
Lentiviral production using lipofectamine 3000 reagent. Add the lipofectamine reagent dropwise and mix by swirling the tip or gently. However, we have not done a comparison for lentiviral production. This protocol is to produce lentivirus stocks from hairpinplko. The recombinant viral vector is then replicated and assembled into complete, pseudotyped virus. The virapower bsd lentiviral support kit includes the following components. How much lipofectamine plasmid is required for lentivirus production. Generation of hightiter lentivirus for the production of transgenic. Lipofectamine 2000 reagent thermo fisher scientific. Designed to efficiently package most thirdgeneration lentivectors, the ppackh1 hiv lentivector packaging kit makes it easy to deliver and express lentiviral constructs in a widerange of mammalian cells. Lentiviral vector production for cell and gene therapy. Production of lentiviral vectors for transducing cells from the central nervous system mingjie li 1, nada husic 1, ying lin 1, b. A lentiviral construct containing the gene of interest along with lentiviral packaging mix is cotransfected into 293t or 293ft cells using lipofectamine 3000 reagent. This procedure can be modified for alternative packaging cell lines or transfection reagents.
More copy number of the integrated gene via lentivirus based method than that via dna transfection method results in significantly improved efficiency of changing protein expression level. Lentiviral production using xtremegene hp transfection reagent. We recommend switching to a different brand of fbs if 293t cells disattach from plates during lentiviral production. User manual a limiteduse label license covers this product. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. After 3648 hrs, harvest all 10 ml medium in a 15 ml. Important tips take care to store lipofectamine 2000 at 4c. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Adherent lentiviral production reagents lipofectamine 3000 transfection reagent 0. Virabind lentivirus concentration and purification kit. Simplified production and concentration of lentiviral vectors.
Once produced, lentivirus can be used for a variety. Production of lentiviral vectors pubmed central pmc. Nov 12, 20 final simplified production and concentration of lentivirus. Recombinant lentivirus production protocol creative biogene. Highest transfection efficiency in many cell types and formats e. Other lentiviral production products largerscale lentiviral production lentisuite basic and. Sigmas universal transfection reagent is a unique formulation of a proprietary polymer blend used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hardtotransfect primary cells.
Hiv cmvegfp virus was produced in hek 293ft cells using either capo 4, lipofectamine 2000 or transitlenti transfection reagent per the manufacturers protocol. Optimization of lentiviral vector production using. Does anyone have experience with the lipofectamine 2000. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. Lentiviral production using xtremegene hp transfection reagent during virus production, low titers and toxicity can be challenging. Lipofectamine 2000 reagent for highefficiency transfection of 293ft producer cell line. The virapower lentiviral support kits includes the virapower packaging mix, lipofectamine 2000, and a selection agent. The lenti293t cell line has been tested as a suitable cell line for lentiviral production due to high transfection efficiency, high level of protein yield and high titer of lentiviral production. It is advisable to screen multiple lots for efficiency of virus production. Hi everyone, im relatively new to viral transduction, and im looking to see if anyone has experience with lipofectamine 2000 based lentivirus production in 293t cells who can assist me. Mission lentiviral packaging mix technical bulletin. Protocol 2 producing lentivirus in hek293t cells using a.
More copy number of the integrated gene via lentivirus based method than that via dna transfection method. How much lipofectamine plasmid is required for lentivirus. To facilitate packaging, sbi offers a 293tn producer cell line that was optimized for effective production of a high. Protip there can be batch to batch variation when making the pei working stock, therefore the ratio of. Here, we demonstrate the effectiveness of lipofectamine 3000 reagent for the production of highquality, hightiter lentivirus while minimizing time and reagent usage. Comparison of capo 4, lipofectamine 2000 or transitlenti generated lentivirus. Aug 07, 2012 add 2 microliters of lipofectamine ltx reagent to tube 1, 3 microliters to tube 2, 4 microliters to tube 3 and 5 microliters to tube 4. Lentiviral tropism is determined by the ability of the viral envelope protein to interact with receptors at the host cell surface. Lentiviral production using xtremegene hp transfection.
The aim of the present study was to optimize the polyethylenimine peimediated transfection method in order to simplify the efficient production of lentiviral vectors lvvs, and to compare the capo 4 and peimediated transfection methods for producing lvvs. Once a batch of pei is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Lentivirus production was optimised in small scale experiments by transfections in 24 well plates. Lv production in adherent cells with lipofectamine 3000 reagent. Lipofectamine 2000 transfection reagent invitrogen. Retroviral vector production right occurs in a similar manner to lentivirus production, with the main difference being that the initial step is transfection with a single plasmid.
Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Comments are turned off autoplay when autoplay is enabled, a suggested video will automatically play next. Protocol compatible with serum, serumfree medium and antibiotics. Protocol 2 producing lentivirus in hek293t cells using a 2nd generation lentiviral system before any work begins, you must have contacted your institutions biosafety office to receive permission and institutionspecific instructions on working with lentivirus. This easytouse protocol uses xtremegene hp transfection reagent for fast and robust lentiviral preparation, with high virus titers being suitable to successfully transduct target cells. Universal transfection reagent lentivirus generation in. Lentivirus was collected 48 hours posttransfection and concentrated by prolonged centrifugation. Virabind lentivirus concentration and purification kit provides an efficient system for quick lentiviral purification with high recovery 60%. Transitlenti transfection reagent is designed to enhance delivery of packaging and transfer vectors to adherent hek 293t and suspension 293f cell types to increase recombinant lentivirus production. Infection the target cells with lentivirus, the cell culture media can. Description based on our proprietary polymer synthesis technology, polyjet dna in vitro transfection reagent is formulated as a biodegradable polymer based dna transfection reagent that ensures effective and reproducible transfection on hek293, cos7, nih3t3, hela, cho and a broad ranges of hardtotransfect. Different titration methods of lvv stocks, as well as different culture media, culture durations, cell. Joy snider 1 1 department of neurology and hope center for neurological disorders, washington university school of medicine.
Invitrogen virapower bsd lentiviral support kit 20. Animation describing transfection of plasmid dna into cells. Incubate the cells for 18 h, or until the following morning. Lentivirus based stable cell line generation syd labs.
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