2.3. Lentiviral Vector Production and Cell Transduction

Lenti-X 293T cells (Clontech^®, Takara Bio Europe SAS, Saint-Germain-en-Laye, France) were co-transfected together with lentiviral gene transfer plasmids (U6-gRNA:hPGK-puro-2A-tBFP or pLV LoxP-EF1A espCas9(ns)-T2A-mCherry-LoxP) and packaging plasmids (pSPAX2 (Addgene plasmid # 12260) and a VSV-G encoding plasmids) [22]. Lentiviral supernatants were collected 48 h, 72 h and 96 h post transfection, filtrated and concentrated 100× by ultracentrifugation. Lentiviral vectors were then titrated with qPCR Lentivirus Titration (Titer) Kit (ABM^®, LV900, Richmond, BC, Canada) and used to transduce LP-1 cells.

Cells were double transduced with CRISPR lentiviral vectors and lentiviral guide RNA for CD38 using 30 lentiviral particles per cells. After 72 h, cells expressing mCherry and BFP were isolated by FACS (BD Biosciences, San Jose, CA, USA) and then maintained in culture. Cells that were not expressing CD38 receptor anymore were selected by FACS using the APC-conjugated CD38 mAb (HIT2, BioLegend, San Diego, CA, USA). This step was done twice and the KO for CD38 was confirmed by Western blot.