Pspaxii

Manufactured by Addgene

PsPAXII is a lentiviral packaging plasmid used for the production of lentiviral particles. It encodes the necessary viral structural and packaging proteins required for the assembly of lentiviral particles. PsPAXII does not contain the viral genome and cannot produce infectious viral particles on its own.

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Lab products found in correlation

Alliance hiv 1 p24 antigen elisa kit, by PerkinElmer (1 mentions) Q5 polymerase, by Thermo Fisher Scientific (1 mentions) Phage cmv luc2 ires zsgreen, by BEI Resources (1 mentions) Phdm hgpm2, by BEI Resources (1 mentions) Prc cmv rev1b, by BEI Resources (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)

2 protocols using pspaxii

SARS-CoV-2 Spike Lentiviral Production

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Lentiviral particles encoding the SARS-CoV-2 spike were prepared by transient transfection of HEK293Tn cells using the CaCl2 method. The lentiviral vector pCDH-EF1a-GFP (System Bioscience), the packaging plasmid psPAXII (Addgene), the spike expression vector phCMV-SARS-CoV-2-Spike (a gift from O. Schwartz), and the pRev plasmid (a gift from P. Charneau) were mixed at a 2:2:1:1 ratio and transfected at 252 µg DNA per 175 cm² cell flask. The pQCXIP-Empty plasmid was used as a negative control for spike expression. At 48 h after transfection, supernatants were collected and concentrated by ultracentrifugation at 23,000 × g for 1 h 30 m at 4 °C on a 20% sucrose cushion. Viral particles were resuspended in PBS and frozen in aliquots at −80 °C until use. Gag p24 antigen concentration was measured with the Alliance HIV-1 p24 Antigen ELISA kit (Perkin Elmer).
Point mutations to generate the mutants were introduced by site-directed mutagenesis of phCMV-SARS-CoV-2-Spike using Q5 polymerase (Thermo Scientific) and validated by sanger sequencing. The primers used are listed in the supplementary information.

Meyer B., Chiaravalli J., Gellenoncourt S., Brownridge P., Bryne D.P., Daly L.A., Grauslys A., Walter M., Agou F., Chakrabarti L.A., Craik C.S., Eyers C.E., Eyers P.A., Gambin Y., Jones A.R., Sierecki E., Verdin E., Vignuzzi M, & Emmott E. (2021). Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential. Nature Communications, 12, 5553.

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SARS-CoV-2 Spike Mutation Analysis Protocol

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All the spike mutations were inserted into a codon-optimized version of the Wuhan-Hu-1 SARS-CoV-2 spike (GenBank accession no. QHD43416.1) cloned into a phCMV backbone (GenBank accession no. AJ318514). Mutations were introduced into the phCMV-Spike plasmids using mutagenic primers and the Q5 site-directed mutagenesis kit (New England Biolabs [NEB]). In addition, a truncation of 19 amino acids was introduced into the cytoplasmic tail of each spike studied (deletion at amino acids 1255 to 1273 in the Wuhan-Hu-1 spike) to promote spike incorporation into lentiviral particles, as described previously (67 (link), 68 (link)). All the mutant plasmids were sequenced prior to use. Primers used for mutagenesis and sequencing are reported in Table S1. The plasmid pQCXIP-empty was used as a negative control for spike expression (50 (link)). Plasmids used to produce GFP-expressing lentiviruses were the lentivector backbone pCDH-EF1α-GFP (System Biosciences), the packaging plasmid psPAXII (Addgene), and the pRev plasmid (a gift from P. Charneau). For the production of luciferase-expressing lentivirus, the plasmids used were pHAGE-CMV-Luc2-IRES-ZsGreen (NR-52516), pHDM-Hgpm2 (NR-52517), pHDM-tat1b (NR-52518), pRC-CMV-rev1b (NR-52519), all generated in J. Bloom’s laboratory (69 (link)) and obtained from BEI Resources (kit NR-53816).

Gellenoncourt S., Saunders N., Robinot R., Auguste L., Rajah M.M., Kervevan J., Jeger-Madiot R., Staropoli I., Planchais C., Mouquet H., Buchrieser J., Schwartz O, & Chakrabarti L.A. (2022). The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants. Journal of Virology, 96(19), e01301-22.

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