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Workflow for pseudovirus production using Lenti-X Packaging Single Shots (Envelope-Free)

Workflow for pseudovirus production using Lenti-X Packaging Single Shots (Envelope-Free).

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631294: Lenti-X Packaging Single Shots (Envelope-Free)

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

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

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

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. 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 from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 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. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and 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 since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

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 pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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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 ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. 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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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of 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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632668: Lenti-X SARS-CoV-2 Packaging Single Shots (WT Spike, Full Length)

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

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

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

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. 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 from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 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. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and 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 since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

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 pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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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 ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. 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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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of 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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632669: Lenti-X SARS-CoV-2 Packaging Single Shots (D614G Spike, Full Length)

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

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

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

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. 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 from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 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. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and 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 since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

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 pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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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 ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. 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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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of 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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632670: Lenti-X SARS-CoV-2 Packaging Single Shots (WT Spike, Truncated)

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

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

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

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. 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 from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 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. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and 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 since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

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 pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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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 ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. 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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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of 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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632671: Lenti-X SARS-CoV-2 Packaging Single Shots (D614G Spike, Truncated)

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631292: Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Full Length)

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631293: Lenti-X SARS-CoV-2 Packaging Single Shots (B.1.351 Spike, Truncated)

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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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The AAVpro 293T Cell Line produces high titers of adenoassociated virus (AAV)

The AAVpro 293T Cell Line produces high titers of adenoassociated virus (AAV). AAV2-pseudotyped virus was generated using the AAVpro Helper Free System (AAV2) (Cat. # 6230) with three different packaging cell lines. The AAVpro 293T Cell Line produced greater or equivalent titers to the commonly used 293T/17 clone, and greater titers than AAV-293, a subclone of HEK 293 selected for AAV production.

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

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

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

Transduction of an ACE2-positive cell line using pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots. Panel A. 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 from SARS-CoV-2, respectively, and encoding the fluorescent protein ZsGreen1. Experiments were performed in duplicate. 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. Following the same protocol, pseudoviruses encoding firefly luciferase were produced in duplicates and 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 since spike-pseudotyped viruses are incapable of infecting HEK293T cells in the absence of ACE2). Resulting levels of luciferase were measured 6 days posttransduction.

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Functional titers obtained using Lenti-X SARS-CoV-2 Packaging Single Shots

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 pseudovirus produced using Lenti-X SARS-CoV-2 Packaging Single Shots (WT or D614G spike, Truncated). Panel B. Corresponding microscopy images of ACE2 HEK293T cells transduced with SARS-CoV-2 pseudoviruses encoding the ZsGreen1 reporter at 72 hours post-infection.

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632673: Lenti-X SARS-CoV-2 Packaging Mix (D614G Spike, Full Length)

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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 ACE2 protein as a blocking agent

Neutralizing activity of soluble ACE2 protein against SARS-CoV-2 pseudoviruses bearing truncated WT or D614G variants of the spike protein. 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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Neutralization data with convalescent serum

Neutralizing activity of pooled convalescent serum from SARS-CoV-2-infected non-human primates against SARS-CoV-2 pseudovirus. WT (truncated) SARS-CoV-2 pseudovirus was produced using Lenti-X SARS-CoV-2 Packaging Single Shots, incubated with serial dilutions of 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.