Pcmv vsv g envelope vector

Human HER2 cDNA (RC212583; Origene) was subcloned into pMXs-IRES-Blasticidin (Cell Biolabs). HER2 mutants were generated by site-directed mutagenesis. Retroviral expression vector retrovirus was produced by transient transfection of HEK 293T cells with the retroviral ERBB expression vector pMXs-IRES-Blasticidin, pCMV-Gag-Pol vector, and pCMV-VSV-G-Envelope vector (Cell Biolabs). Briefly, HEK 293T cells were plated (2.5 × 10⁵ per plate) in 6-well plates (Corning) and incubated overnight. The next day, retroviral plasmids (1 µg of ERBB, 0.32 µg of pCMV-Gag-Pol, and 0.16 µg pCMV-VSV-G) were mixed in 150 µL of Opti-MEM (Life Technologies). The mixture was incubated at room temperature for 5 minutes and then added to Opti-MEM containing transfection reagent Lipofectamine (Invitrogen) and incubated for 20 minutes. Mixture was then added dropwise to HEK 293T cells. The next day, the medium was replaced with fresh culture medium, and retrovirus was harvested at 24 hours.

K562 cells stably expressing a dCas9-KRAB fusion protein were kindly provided by Jonathan S. Weissman.¹⁹ (link) pU6-sgRNA-EF1Alpha-puro-T2A-BFP (Addgene, Plasmid #60955) was used to express sgKAT and sgNC sgRNAs; and pU6-sgRNA-EF1Alpha-BSR-T2A-BFP (puromycin-resistance gene replaced by blasticidin S-resistance gene) was used to express sgSIRT5 sgRNAs. The generation of stably gene knockdown K562 cells was performed using a CRISPRi system.¹⁹ (link) Briefly, double-stranded sgRNA oligos (Table S1) were synthesized and inserted into backbone plasmids using BstXI and BlpI restriction sites. Lentivirus expressing sgRNAs were packaged and produced using HEK293T cells with pCMV-VSV-G envelope vector (Cell Biolabs, Cat. # RV-110) and pCMV-dR8.91 packaging vector (Creative Biogen, Cat. #OVT2971). K562-dCas9-KRAB cells stably expressing sgRNAs were generated upon lentiviral infection, and selected using corresponding antibiotics and fluorescence-activated cell sorting of BFP-positive cells.