Plvx puro

Neu5Gc was expressed on the surface of modified human erythrocytes by expressing chimpanzee CMAH in ex-vivo-cultured red blood cells (cRBCs)^{26 (link)}. Briefly, differentiation and proliferation of bone marrow-derived CD34⁺ hematopoietic stem cells (Lonza) were achieved in Iscove’s Modified Dulbecco-based medium (Biochrom) supplemented with 5% solvent/detergent virus-inactivated human plasma (Octaplas, Octapharma), stem cell factor (R&D Systems), hydrocortisone (Invitrogen), IL-3 (R&D Systems), and erythropoietin (Amgen) according to a published protocol^{48 (link)} with the following modifications. Cells were transduced on day 7 with lentivirus harbouring either the chimpanzee (Pan troglodytes) CMAH cDNA sequence in pLVX-Puro (Clontech) or the empty vector, pLVX-Puro. The CMAH insert was codon-optimized for human expression and synthesized with a C-terminal tobacco etch virus (TEV) cleavage site and FLAG-c-myc tags (GeneArt). Transductants were selected on 2 mg ml⁻¹ puromycin (Sigma–Aldrich). Drug selection was maintained until day 13 when cells were co-cultured on a murine MS-5 stromal cell layer. Cells were replated on MS-5 stroma on day 18 and harvested on day 20 or 21. Cells were stored at 4 °C in incomplete RPMI (RPMI-1640 [Sigma–Aldrich] supplemented with 25 mM HEPES and 50 mg l⁻¹ hypoxanthine) until binding and flow cytometry assays.

Viral particles were produced using Lenti-X 293T cells. Briefly, 3x10⁶ Lenti-X-293T cells were seeded one day before transfection. For the transfection assays, Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used according to the manufacturer's protocol and utilizing a mix of Lenti-X HT packaging systems (4 µg) and 1 µg of one of the following lentiviral vectors, pLVX-Puro (Takara Bio, Inc.) or the pLVX-EF1alpha-SARS-CoV-2-ORF3a-2xStrep-IRES-Puro vector (cat no. 141383; Addgene, Inc.) (43 (link)). After 48 h, to eliminate detached cells and cell detritus, the medium containing the viral particles was filtered through a 0.45-µm polyethersulfone membrane (Merck KGaA). The titer of viral particles was tested using Lenti-X GoStix (Takara Bio, Inc.). The medium containing the viral particles was aliquoted and stored at -80˚C until use. For the transduction assays, 1x10⁶ target cells were seeded one day before transduction. The target cells were transduced with 200 µl medium containing lentiviral particles (~1x10⁶ Inclusion-Forming Units). To obtain stable cell lines, after 72 h of transduction, the cells were incubated with 0.5 µg/ml puromycin for 3 weeks. The medium containing puromycin was replaced every three days; stable cells were cultured under normal conditions without puromycin. Cells were always cultivated or treated at 37˚C in a 5% CO₂ atmosphere.