pLVX-IRES-mCherry Vector

Description

The pLVX-IRES-mCherry Vector is a bicistronic lentiviral expression vector that can be used to generate high-titer lentivirus for transducing dividing or nondividing mammalian cells. The vector contains an internal ribosomal entry site IRES which allows a gene-of-interest and the mCherry fluorescent protein to be simultaneously coexpressed from a single mRNA transcript. When used with Lenti-X Packaging Single Shots and the Lenti-X 293T Cell Line Cat. No. 632180 , the vector generates high titers of replication-incompetent, VSV-G-pseudotyped lentivirus. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back Back Lentiviral vectors with fluorescent proteins.Lenti-X vectors contain sequence elements that facilitate lentiviral packaging and/or boost transgene expression, including the LTRs, packaging signal , Rev response element RRE , and central polypurine tract/central termination sequence cPPT/CTS from HIV-1; and the woodchuck hepatitis virus post-transcriptional regulatory element WPRE . Vectors can express your protein fused at its N- or C- terminus to either a green AcGFP1 or red DsRed-Monomer fluorescent protein tag, or coexpress it as a separate protein along with ZsGreen1 shown , mCherry, or tdTomato. Required Products License Statement Lenti-X Packaging Single Shots VSV-G provide an extremely simple and consistent one-step method for producing high-titer lentivirus. No additional transfection reagent is needed because Lenti-X Packaging Single Shots VSV-G consist of pre-aliquoted, lyophilized, single tubes of XfectTransfection Reagent premixed with an optimized formulation of VSV-G pseudotyped Lenti-X lentiviral packaging plasmids. High-titer virus is produced by simply reconstituting this mixture with your lentiviral vector of choice in sterile water and adding it to 293T cells, e.g., Lenti-X 293T Cells Cat. # 632180 , in a 10 cm dish. Back Back Consistent, high-efficiency transfections lead to high titers.A lentiviral vector containing the ZsGreen1 gene was packaged according to the Lenti-X single shots protocol in four independent experiments. Briefly, 7 mug of pLVX-ZsGreen1 plasmid was added to each of four Lenti-X single shots; the tubes were vortexed for 20 sec and incubated at room temperature for 10 min. Then, the mixture was added to cultured Lenti-X 293T cells that were approximately 80 % confluent. 48 hours after transfection, the cells were imaged by fluorescence microscopy Panel A, top and light microscopy Panel A, bottom . After images were taken, the supernatant was harvested and used infect HT1080 cells for titer determination Panel B, IFU/ml . Back High-titer virus was produced regardless of the lentiviral vector backbone with Lenti-X packaging single shots.A CMV ZsGreen1 expression cassette was cloned into several lentiviral vector backbones. These vectors were then packaged into lentivirus using the Lenti-X packaging single shots following the provided protocol. Briefly, 7 mug of pLVX-ZsGreen1 plasmid was added to each of four Lenti-X single shots, and the tubes were vortexed for 20 sec and incubated at room temperature for 10 min. Then, the mixture was added to cultured Lenti-X 293T cells that were approximately 80 % confluent. After 48 hours, titer was determined using several methods. To determine infectivity, the supernatant was harvested and used to infect HT1080 cells Flow Cytometry . Harvested viral supernatants were also analyzed by RT-PCR to quantify viral genome copies qRT-PCR, Lenti-X qRT-PCR Titration Kit , ELISA to measure p24 p24 ELISA, Lenti-X p24 Rapid Titer Kit , and by a rapid lentiviral detection method Lenti-X GoStix . Back A comparison of fourth- and third-generation lentiviral packaging systems .Our Lenti-X Packaging single shots utilize a packaging system that consists of five separate components Panel A , mixed in proprietary proportions for optimized packaging activity. The separation of thegag,pol, andenvgenes effectively reduces the incidence of RCL Wuet al., 2000 . High levels of expression of essential viral components are driven by the Tet-Off and Tat transactivators, which induce a cascade of expression that results in high titers of lentivirus. Thepolgene is fused tovprto ensure transport of the reverse transcriptase/integrase protein into the recombinant lentiviral particle. Not all vector elements are shown. Other 3rd generation lentiviral packaging systems Panel B generate lower titers, do not contain separategagandpolsequences, and do not use a transactivation cascade mechanism. Back The Lenti-X Concentrator is a complete reagent for the concentration of infectious lentiviral vector particles. This reagent provides a scalable alternative to ultracentrifugation for viral particle concentration. Vector supernatants can be concentrated 10100 fold, depending upon the volumes used. Back Back Back Concentrate lentivirus from any volume or from any lentiviral titer.Lentiviral supernatant was diluted into 250 ml and then concentrated down to 2.5 ml using Lenti-X Concentrator Panel A . 10-fold serial dilutions of a high-titer lentiviral supernatant high, medium, and low were concentrated from a volume of 10 ml down to 100 mul using the Lenti-X Concentrator Reagent Panel B . Titrations were performed using HT1080 cells and flow cytometry 48 hr post-transduction. Back