Streamlined production of high-titer lentivirus in a 96-well format
Production of lentiviral vectors in a 96-well format is a common starting point for a variety of high-throughput applications, including arrayed CRISPR or shRNA-based screening and vector optimization workflows. However, obtaining sufficient viral titers in a high-throughput context can prove challenging, as production processes typically involve optimizing proportions of the respective packaging reagents and distributing them in minute quantities. To help researchers overcome these obstacles and streamline their process development efforts, we have adapted our popular Lenti-X packaging system to enable consistent high-titer production of VSV-G-pseudotyped lentiviral particles in 96-well plates. Lenti-X Packaging Single Shots (96-well, VSV-G) consist of a lyophilized mixture of Xfect Transfection Reagent and optimized ratios of Lenti-X packaging plasmids, pre-aliquoted in a 96-well format, providing researchers with the following benefits:
Provided with each kit are four 96-well plates that come individually packaged in foil pouches. The plates contain a lyophilized mixture of Xfect Transfection Reagent and Lenti-X packaging plasmids that has been precisely aliquoted into each well. The plates are sealed with a pierceable foil that allows for processing on automated liquid handling platforms.
Lenti-X Packaging Single Shots 96-well Plate (VSV-G).
Lenti-X Packaging Single Shots (96-well, VSV-G) provide a streamlined method for high-throughput lentivirus production and are compatible with all commonly available lentiviral transfer vector backbones. Users simply add 10 µl of plasmid encoding their transfer vector of choice to each well and mix to solubilize the pre-aliquoted transfection mixes. The resulting nanoparticle complexes are then used to transfect packaging cells (e.g., Lenti-X 293T cells) in a separate plate. After 48–72 hours, high-titer lentivirus can be harvested from the supernatant in each well and used to transduce target cells in a separate plate. This workflow is summarized by the following figure:
Protocol for Lenti-X Packaging Single Shots 96-well Plate (VSV-G).
Lenti-X Packaging Single Shots (96-well, VSV-G) employ Takara Bio's proprietary fourth-generation lentiviral packaging system, which has been extensively optimized to maximize resulting titers. In a 96-well context, this system typically yields titers of 1 x 107–1 x 108 IFU/ml, as demonstrated by the data below.
Reliable high-titer lentivirus production with Lenti-X Packaging Single Shots (96-well, VSV-G). A transfer vector plasmid encoding ZsGreen1 was added to each well of a Lenti-X Packaging Single Shots 96-well Plate (VSV-G) as specified in the product user manual, and solubilized transfection mixes were then applied to Lenti-X 293T cells in a separate plate. Following overnight incubation at 37°C, transfection of producer cells from four randomly selected wells was confirmed by fluorescence microscopy (Panel A). 48 hours post-transfection, serially diluted lentiviral supernatants were applied to 293T target cells in a separate plate in the presence of polybrene (6 µg/ml). Transduced cells were analyzed by flow cytometry 72 hours post-transduction, and infectious unit titers were calculated using target cell populations that were transduced at <10% efficiency to ensure that there was no more than one copy of the transgene per cell (Panel B). Resulting titers were ≥1 x 107 IFU/ml for each of the four randomly selected wells.
To effectively support high-throughput workflows and avoid the introduction of experimental artifacts, a product must provide consistent performance from well to well and plate to plate. To ensure the reliability of Lenti-X Packaging Single Shots (96-well, VSV-G) for high-throughput lentivirus production, well-to-well variation in transfection efficiencies was analyzed using plates from two different production lots.
Consistent transfection efficiencies from well to well and lot to lot with Lenti-X Packaging Single Shots (96-well, VSV-G). Transfection mixes were prepared using a transfer vector plasmid encoding ZsGreen1 and two different lots of Lenti-X Packaging Single Shots (96-well, VSV-G). The transfection mixes were applied to Lenti-X 293T cells in a separate plate and, following an overnight incubation, GFP expression was analyzed using a fluorescence plate reader (Panel A; amounts of plasmid applied to each well are indicated in ng). High, low, and average levels of GFP expression were determined for cells transfected with 40 ng of plasmid (wells indicated by black box in Panel A) using each lot and the standard deviation and coefficient of variation among wells from each lot were calculated (Panel B; n = 72 for each lot). As shown, average levels of GFP expression were comparable between lots, and the well-to-well coefficient of variation (CV) was less than 15% for each lot.
Given the reality that quantitation and adjustment of transfer vector plasmid concentrations may not be practical in the context of a high-throughput workflow, it is helpful to have a production system that still yields usable titers despite variations in this regard. With this in mind, viral titer production with Lenti-X Packaging Single Shots (96-well, VSV-G) was evaluated across a range of transfer vector plasmid input amounts.
Usable viral titers from varying inputs of transfer vector plasmid DNA. Transfection mixes were prepared using Lenti-X Packaging Single Shots (96-well, VSV-G) and 0–160 ng inputs of transfer vector plasmid before being applied to Lenti-X 293T producer cells. Resulting transfection efficiencies were analyzed by fluorescence microscopy (Panel A). Viral titers obtained for the indicated range of transfer vector input amounts were measured by qRT-PCR using the Lenti-X qRT-PCR Titration Kit (Panel B). As shown, usable viral titers were obtained across a wide range of transfer vector input amounts.
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