Lenti-X Packaging Single Shots

The Lenti-X Packaging Single Shots (VSV-G) protocol

The Lenti-X Packaging Single Shots (VSV-G) protocol.

Consistent, high-efficiency transfections lead to high titers

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 µg 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).

High-titer virus was produced regardless of the lentiviral vector backbone with Lenti-X packaging single shots

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 µg 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).

A comparison of fourth- and third-generation lentiviral packaging systems

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 the gag, pol, and env genes effectively reduces the incidence of RCL (Wu et 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. The pol gene is fused to vpr to 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 separate gag and pol sequences, and do not use a transactivation cascade mechanism.