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Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

Workflow for pseudovirus production using Lenti-X SARS-CoV-2 Packaging Single Shots (Universal).

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Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

Transduction of an ACE2-positive cell line with SARS-CoV-2 pseudovirus. Lenti-X Packaging Single Shots (Universal) was used to produce truncated SARS-CoV-2 spike B.1.1.7 pseudovirus encoding the fluorescent protein ZsGreen (Panel A) or SARS-CoV-2 spike B.1.1.7 pseudovirus encoding firefly luciferase (Panel B). Panel A. Concentrated supernatant (20X; 30 or 10 µl) was used to transduce a HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 µg/ml polybrene in 48-well plates. The transduction efficiencies for each sample were measured by flow cytometry 6 days post-transduction. The percentage of ZsGreen-positive (ZsGreen+) cells and mean fluorescence intensity (MFI) are shown. Panel B. Concentrated supernatant (20X; 30, 20, or 10 µl) was used to transduce a HEK293T cell line stably expressing the human ACE2 receptor in the presence of 6 µg/ml polybrene in 48-well plates. HEK293T cells lacking the ACE2 transgene were transduced to determine background luminescence levels (NC; negative control). Luminescence values for each sample were measured 6 days post-transduction.

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Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) uses Takara Bio’s Lenti-X 4th-generation packaging system

Lenti-X Packaging Single Shots (Envelope-Free) uses Takara Bio’s Lenti-X 4th-generation packaging system. Panel A. Plasmids expressing all viral components except the SARS-CoV-2 spike protein that are contained in lyophilized Single Shots formulation. High expression of essential viral components are driven by the Tet-Off and Tat transactivators. In addition, the gag and pol genes are separated and the pol gene is fused to vpr to ensure transport of the reverse transcriptase/integrase protein into the recombinant viral particle. Panel B. The remaining viral component, the desired SARS-CoV-2 spike variant mutant is cloned into the pEmpty Vector and used in the single shots reaction. Expression of the spike variant mutant is driven by a CMV promoter.

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631295: Lenti-X SARS-CoV-2 Packaging Single Shots (Universal)

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632180: Lenti-X 293T Cell Line

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Clontech's lentiviral packaging system (Panel A) and a lentiviral packaging system from a leading competitor (Panel B) were each used to generate viral supernatants from their respective lentiviral system vectors that were engineered to express the ZsGreen1 fluorescent protein

Clontech's lentiviral packaging system (Panel A) and a lentiviral packaging system from a leading competitor (Panel B) were each used to generate viral supernatants from their respective lentiviral system vectors that were engineered to express the ZsGreen1 fluorescent protein. As little as 10 µl of supernatant from Lenti-X transduced the majority of these HeLa cells, whereas 10 µl of supernatant from the other system transduced only a small percentage of the cells. Transduced cells were quantified by flow cytometry.

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Transduction of neural progenitor cells by Lenti-X lentivirus

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).

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High-titer lentivirus production

High-titer lentivirus production. Lenti-X 293T cells were transduced with the indicated volumes (µl) 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.

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293T cell line for higher titers

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 lines—producing over 6X more virus than 293FT cells and up to 30X more virus than the parental HEK 293 cell line.

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Analysis of ACE2 expression by flow cytometry

Analysis of ACE2 expression by flow cytometry. ACE2 expression at the cell surface of the Human ACE2 293T Cell Line was detected by flow cytometry using an Alexa Fluor 488-conjugated monoclonal anti-ACE2 antibody (R&D Systems, Cat. # FAB9332G). Non-labelled cells were used as a negative control.

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Confirmation of stable ACE2 expression across multiple cell passages

Confirmation of stable ACE2 expression across multiple cell passages. Relative mRNA expression levels of ACE2 across successive passages of the Human ACE2 293T Cell Line were determined by RT-qPCR using the One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Cat. # RR064A). Ct values for ACE2 expression were normalized to Ct values for the housekeeping gene ACTB in triplicates. ACE2 expression in the stable cell line was determined relative to a 293T cell line by calculating 2^(–ΔΔCt).

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding ZsGreen1

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding ZsGreen1. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (truncated form) and encoding the fluorescent protein ZsGreen1. 100 μl of supernatant from each prep was used to transduce the Human ACE2 293T Cell Line. Panel A. Microscopy images of transduced cells at 72 hours post-infection. Panel B. Transduction efficiencies for each sample were measured by flow cytometry 6 days post-transduction.

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (full length or truncated forms) and encoding the firefly luciferase protein. 10 μl of concentrated virus (21X) from each prep was used to transduce the Human ACE2 293T Cell Line. Luciferase activity was measured 6 days post-transduction using a control virus lacking an envelope protein to provide a background signal for analysis of the luciferase activity.

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Neutralization data with convalescent serum

Use of the Human ACE2 293T Cell Line in neutralization assays employing pooled non-human primate convalescent serum to SARS-CoV-2 as a blocking agent. WT (truncated) SARS-CoV-2 pseudovirus was incubated with serial dilutions of the convalescent serum (BEI Resources, Cat. # NR-52401) and applied to the Human ACE2 293T Cell Line. 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. Negative control was performed using serum from healthy macaques.

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Neutralization data with ACE2 protein as a blocking agent

Use of the Human ACE2 293T Cell Line in neutralization assays employing soluble ACE2 protein as a blocking agent. 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 the Human ACE2 293T Cell Line. 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.

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631289: Human ACE2 293T Cell Line