Takara Lenti-X SARS-CoV-2 Packaging Single Shots (D614G Spike, Full Length) \12 Rxns

Lenti-X SARS-CoV-2 Packaging Single Shots (D614G Spike, Full Length) enable streamlined production of high-titer lentivirus pseudotyped with the spike protein from SARS-CoV-2 (D614G mutant) by providing pre-aliquoted, lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimized formulation of Lenti-X lentiviral packaging plasmids. Pseudovirus production simply involves reconstituting a tube of lyophilized packaging mix with a lentiviral transfer vector of choice in sterile water and adding the mixture to 293T cells (e.g.,Lenti-X 293T Cells, Cat. # 632180) in a 10-cm dish.

Each kit includes 12 tubes of Lenti-X SARS-CoV-2 Packaging Mix (D614G Spike, Full Length; Cat. # 632673), 6 tubes of Lenti-X SARS-CoV-2 Packaging Mix (No-Envelope Control; Cat. # 632676) for production of pseudoviral particles lacking an envelope protein, and the Lenti-X SARS-CoV-2 Vector Set (Cat. # 632677), which includes two self-inactivating lentiviral transfer vectors encoding ZsGreen1 and firefly luciferase, respectively, under the CMV promoter, to be used as reporters for viral transduction.

For your convenience, the packaging mixes included in the kit are also sold separately.

Workflow for pseudovirus production using 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.

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.

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.

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.

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.