pLVX-EF1alpha-IRES-mCherry Vector

Description

pLVX-EF1alpha-IRES-mCherry is a bicistronic lentiviral expression vector that can be used to generate high-titer lentivirus for transducing virtually any dividing or nondividing mammalian cell type, including primary and stem cells. The vector contains an internal ribosomal entry site IRES that allows a gene-of-interest and the red fluorescent protein mCherry to be simultaneously coexpressed from a single mRNA transcript. Expression of the transcript is driven by the constitutively active human elongation factor 1 alpha EF1alpha promoter. 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 Expression of AcGFP1 driven by the EF-1 alpha promoter in stem cell lines is higher than expression driven by the CMV promoter.The mouse embryonic stem cell lines E14 Panel A and D3 Panel B were transduced by Lenti-X lentivirus, expressing AcGFP1 either under the control of the CMV promoter or the Elongation factor alpha EF-1 alpha promoter. The expression level of AcGFP1 in infected cells five days postinfection was monitored by FACS analysis using the FL1 channel. The expression of AcGFP1 driven by the EF-1 alpha promoter in both stem cell lines was considerably higher compared to the CMV promoter. This is mainly due to a considerably lower rate of silencing of the EF-1 alpha promoter in stem cells compared to the CMV promoter as published Wang, et al. 2008 Stem Cells Dev17:279289 . Back 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 License Statement The Lenti-X 293T Cell Line is a subclone of the transformed human embryonic kidney cell line, HEK 293, which is highly transfectable and supports high levels of viral protein expression. When transfected with Lenti-X Packaging Single Shots and a lentiviral vector, these cells are capable of producing lentiviral titers as high as >108ifu/ml, as determined by flow cytometry. The cell line also constitutively expresses the simian virus 40 SV40 large T antigen. Back High-titer lentivirus production.Lenti-X 293T cells were transduced with the indicated volumes mul of lentiviral packaging supernatant generated with the Lenti-X Expression System and then selected with puromycin for 9 days to allow the formation of the resistant colonies, which were then stained with crystal violet. Back Back Back Transduction of neural progenitor cells by Lenti-X lentivirus.Recombinant lentivirus for expressing ZsGreen1 was produced using Lenti-X virus and used to transduce normal human neural progenitor cells. A single transduced cell is shown under phase contrast microscopy Panel A and fluorescence microscopy Panel B . Back 293T cell line for higher titers.We used our fourth-generation lentiviral packaging system and one of our pLVX-lentiviral vectors to compare the virus production of the Lenti-X 293T Cell Line to that of two other commonly used HEK 293-based cell lines. Lenti-X 293T cells clearly outperformed the other cell linesproducing over 6X more virus than 293FT cells and up to 30X more virus than the parental HEK 293 cell line.