Lenti-XTM Packaging Single Shots (VSV-G)

Description

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