Lentiviral vectors for the expression of SARS-CoV-2 spike (delta variant B.1.617.2) (NCBI accession number OX014251.1 ) and SARS-CoV-2 nucleocapsid (NCBI accession number OP359729.1 ) were designed and synthesized by GenScript, together with two packaging plasmids (pMD2.G and psPAX2). The full-length sequences of spike and Ncap were used. Additionally, the delta spike sequence was modified for codon optimization and the insertion of a 2P mutation for the stabilization of the sequence (31 (link)). To facilitate the trafficking of spike and Ncap to the exosomes, the proteins were linked to the N terminus of the exosome-specific tetraspanin CD9 by a synthetic transmembrane domain and a secretion signal peptide; this allowed the correct membrane localization of spike and the extracellular localization of Ncap. Lentiviral particles for transduction were generated by transfecting HEK 293T cells with pMG.2 (GenScript), psPAX2 (GenScript), and STX-S_pLenti (GenScript) expressing spike or STX-N_pLenti (GenScript) expressing Ncap at a ratio of 5:5:1 using Lipofectamine 3000 according to the manufacturer’s instructions. Spike and Ncap lentiviral particles were collected at 72 h posttransfection and used to transduce 293F parental cells to generate STX-S and STX-N cells, respectively.
Pspax2
Manufactured by GenScript
PsPAX2 is a lentiviral packaging plasmid used for the production of lentiviral particles. It provides the essential viral packaging components for the generation of replication-incompetent lentiviral vectors.
Automatically generated - may contain errors
Lab products found in correlation
Pmd2 g, by GenScript (4 mentions)
Stx s plenti, by GenScript (3 mentions)
Pmg 2, by GenScript (2 mentions)
Stx n plenti, by GenScript (2 mentions)
Lipofectamine 3000, by GenScript (2 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Z01001 10, by GenScript (1 mentions)
Z02922 10, by GenScript (1 mentions)
Gen5 chs 2, by Agilent Technologies (1 mentions)
4 protocols using pspax2
1
Lentiviral Vectors for SARS-CoV-2 Spike and Nucleocapsid
2
Lentiviral Vectors for SARS-CoV-2 Spike and Nucleocapsid
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Lentiviral vectors for the expression of SARS-CoV-2 spike (delta variant B.1.617.2) (NCBI accession number OX014251.1 ) and SARS-CoV-2 nucleocapsid (NCBI accession number OP359729.1 ) were designed and synthesized by GenScript, together with two packaging plasmids (pMD2.G and psPAX2). The full-length sequences of spike and Ncap were used. Additionally, the delta spike sequence was modified for codon optimization and the insertion of a 2P mutation for the stabilization of the sequence (31 (link)). To facilitate the trafficking of spike and Ncap to the exosomes, the proteins were linked to the N terminus of the exosome-specific tetraspanin CD9 by a synthetic transmembrane domain and a secretion signal peptide; this allowed the correct membrane localization of spike and the extracellular localization of Ncap. Lentiviral particles for transduction were generated by transfecting HEK 293T cells with pMG.2 (GenScript), psPAX2 (GenScript), and STX-S_pLenti (GenScript) expressing spike or STX-N_pLenti (GenScript) expressing Ncap at a ratio of 5:5:1 using Lipofectamine 3000 according to the manufacturer’s instructions. Spike and Ncap lentiviral particles were collected at 72 h posttransfection and used to transduce 293F parental cells to generate STX-S and STX-N cells, respectively.
3
Lentiviral Transduction of SARS-CoV-2 Delta Spike
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Lentiviral vectors for expression of SARS-CoV-2 Spike (Delta variant B.1.617.2, NCBI accession # OX014251.1) were designed and synthesized from Genscript together with the two packaging plasmids (pMD2.G and psPAX2). Full length sequence for Spike was used. Additionally, the delta Spike sequence was modified for codon optimization and insertion of 2P mutation for stabilization of the sequence [14 (link)]. To facilitate the trafficking of Spike to the exosomes, the proteins were linked to N-terminal of the exosome specific tetraspanin CD9 by a synthetic transmembrane domain and a secretion signal peptide: this allowed the correct membrane localization of Spike. Lentiviral particles for transduction were generated by transfecting 293T cells with pMG.2 (Genescript), psPAX2 (Genescript) and STX-S_pLenti (Genscript) expressing Spike at a ratio of 5:5:1 using Lipofectamine 3000 according to the manufacturer’s instruction. Spike lentiviral particles were collected at 72 hours post transfection and used to transduce 293F parental cells to generate STX-S respectively.
4
NLRP6 and ERβ Knockdown Effects
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The cells were seeded in 96-well culture plates at appropriate density were first primed with LPS (100 ng/mL) (00497693, Thermo Fisher Scientific) for 4 h, followed by treatment of 3% DSS for 24 h prior to treatment for 48 h with ERB-041 (1 mM) and TNF-a (5 ng/mL) (Z01001-10, Genscript) or IL-1b (10 ng/mL) (Z02922-10, Genscript). The cell activity was tested according to CCK-8 (40203ES76, YEASEN) protocol. The absorbance at 450 nm was obtained by a microplate reader with Gen5 CHS 2.07 software (BioTek, USA).
Generation of knockdown stable cells pLVX-shRNA2 (PT4052-5, Clontech), psPAX2 (PVT2320, Life Science Market) and pMD2.G (PVT2321, Life Science Market) vectors were purchased from Nova Lifetech Limited (Hongkong, China). Lentivirus-mediated shRNA targeting NLRP6 and ERb mRNA sequences (Listed in Table S1) were obtained from Genscript Biotech Corporation.
pLVX-shNLRP6/pLVX-shERb, psPAX2, and pMD2.G were transfected into the HEK239T cells. The supernatant containing lentivirus was harvested after 40 h and then added into the NCM-460 cells. After 24 h, the medium was replaced with fresh medium. 48 h later, cells were cultured in selection medium containing 10 mg/mL puromycin. All these stable transfected cells were tested regularly by western blotting analysis to ensure the efficiency of down-regulation.
Generation of knockdown stable cells pLVX-shRNA2 (PT4052-5, Clontech), psPAX2 (PVT2320, Life Science Market) and pMD2.G (PVT2321, Life Science Market) vectors were purchased from Nova Lifetech Limited (Hongkong, China). Lentivirus-mediated shRNA targeting NLRP6 and ERb mRNA sequences (Listed in Table S1) were obtained from Genscript Biotech Corporation.
pLVX-shNLRP6/pLVX-shERb, psPAX2, and pMD2.G were transfected into the HEK239T cells. The supernatant containing lentivirus was harvested after 40 h and then added into the NCM-460 cells. After 24 h, the medium was replaced with fresh medium. 48 h later, cells were cultured in selection medium containing 10 mg/mL puromycin. All these stable transfected cells were tested regularly by western blotting analysis to ensure the efficiency of down-regulation.
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