Simplified production of SARS-CoV-2-pseudotyped lentivirus
A common objective in developing COVID-19 therapies is to demonstrate inhibition of SARS-CoV-2 infection. Given the safety measures required in handling live SARS-CoV-2, researchers often employ alternative viruses pseudotyped with the SARS-CoV-2 spike protein (Ni et al. 2020, Shang et al. 2020). To support these efforts, we have adapted our Lenti-X packaging system to enable high-titer production of spike-pseudotyped lentiviral particles in a convenient format.
A common objective in developing COVID-19 therapies is to demonstrate inhibition of SARS-CoV-2 infection. Given the safety measures required in handling live SARS-CoV-2, researchers often employ alternative viruses pseudotyped with the SARS-CoV-2 spike protein (Ni et al. 2020, Shang et al. 2020). To support these efforts, we have adapted our Lenti-X packaging system to enable high-titer production of spike-pseudotyped lentiviral particles in a convenient format.
Lenti-X SARS-CoV-2 Packaging Single Shots kits include lyophilized mixtures of Xfect transfection reagent and lentiviral packaging plasmids bearing any of four different spike protein variants, accompanying transfer vectors encoding ZsGreen1 and luciferase reporters, and transfection mixes for production of lentiviral particles lacking an envelope protein (commonly employed as negative controls). High titers of SARS-CoV-2 pseudovirus are obtained by a simple, one-step method: just add water containing your transfer vector of choice to a tube of single shots, and then apply the reconstituted transfection mix to packaging cells (e.g., Lenti-X 293T cells) in a 10-cm dish. Viral supernatants can be harvested 48–72 hours posttransfection.
Packaging systems are available for the production of viral particles bearing your choice of four different SARS-CoV-2 spike protein variants:
- WT (full length)—derived from the Wuhan-Hu-1 isolate (NC_045512.2) of the novel coronavirus SARS-CoV-2, codon-optimized for expression in mammalian cells
- D614G (full length)—includes the D614G mutation in the spike protein coding sequence; the prevalence of this mutation has risen rapidly since the start of the COVID-19 pandemic (Isabel et al. 2020)
- WT (truncated) and D614G (truncated)—truncations of WT spike and D614G spike coding sequences involving removal of 19 amino acids from C-termini; the truncation is associated with increased abundance of spike protein on the resulting particles, and increased particle infectivity (Johnson et al. 2020)
For your convenience, Lenti-X SARS-CoV-2 packaging mixes are also sold separately.
Overview
- High-performance packaging system consistently yields high titers: avoid spending time optimizing reagents, focus on your downstream assays
- Convenient single shots format minimizes hands-on time and likelihood of errors: simply add water containing a transfer plasmid of choice and apply to packaging cells
- Packaging mixes sold separately or as bundles: conserve resources, purchase only what you need
- Packaging mixes available for different SARS-CoV-2 spike protein variants: choose from the following four options:
- WT (full length)—derived from the Wuhan-Hu-1 isolate (NC_045512.2)
- D614G (full length)—coding sequence includes the highly prevalent D614G mutation
- WT (truncated) and D614G (truncated)—truncated coding sequences result in deletion of 19 amino acids from spike C-terminus and are associated with increased particle infectivity
- Controls provided for neutralization assays: use the included packaging mix for production of lentiviral particles lacking an envelope protein
More Information
Streamlined, one-step workflow
Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots.
Efficient transduction of ACE2-positive cells
Transduction of an ACE2-positive cell line with SARS-CoV-2 pseudovirus. Panel A. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT (Wuhan-Hu-1) or D614G variants of the spike protein, and encoding the fluorescent protein ZsGreen1. 100 µl of supernatant from each prep was used to transduce an HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 µg/ml polybrene in 48-well plates. Transduction efficiencies for each sample were measured by flow cytometry 6 days posttransduction. Panel B. Pseudoviruses encoding firefly luciferase were used to transduce both the ACE2-positive cell line and an HEK293T cell line lacking ACE2 expression (included to provide a background signal for analysis of luciferase activity). Luciferase activity was measured 6 days posttransduction.
Consistently high titers
Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. Functional titers determined via analysis of ZsGreen1 reporter expression in ACE2-positive HEK293T cells transduced with SARS-CoV-2 pseudovirus. Panel B. Corresponding microscopy images of transduced ACE2-positive cells at 72 hours post-infection.
Ideal for neutralization studies
Neutralizing activity of soluble ACE2 protein against pseudoviruses bearing SARS-CoV-2 spike protein variants. Panels A and B. Serial dilutions of soluble ACE2 protein fused to the Fc domain from IgG (ACE2-Fc) were applied, along with SARS-CoV-2 pseudovirus, to ACE2 HEK293T cells. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate.
References
Ni, L. et al. Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals. Immunity. 52, 971–977 (2020).
Shang, J. et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci USA. 117, 11727–11734 (2020).
Isabel, S. et al. Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide. Sci Rep. 10, 14031 (2020).
Johnson, MC. et al. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein. J Virol. 94, e01062-20 (2020).
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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