Lenti x packaging single shot

TF-1 cells (#CRL-2003, ATCC; purchased 2018; authentication and Mycoplasma not tested) were cultured in RPMI 1640 medium with 10% FBS and 2 ng/ml recombinant human GM-CSF (R&D Systems). MOLM14 (from D. Gary Gilliand’s lab; authentication and Mycoplasma not tested) cells were cultured in RPMI 1640 medium with 10% FBS. HEK293T (from D. Gary Gilliand’s lab; authentication and Mycoplasma not tested) was cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% FBS. Cells were cultured at 37 with of 5% CO2. Stable overexpression of IDH2R140Q variants in TF-1 cells was conducted by using pLVX-IRES-Hyg vector harboring FLAG-tagged IDH2 R140Q, R140Q/K413R, or R140Q/K413Q. Briefly, to produce lentivirus, each construct was co-transfected into HEK293T cells using Lenti-X™ Packaging Single shots (Clontech) according to the manufacturer’s instructions. Lentivirus-containing supernatant medium was collected 48 hours after transfection, filtered before adding to the indicated host cell lines. 24 hours after infection, target cells were subjected to hygromycin selection (Invitrogen). The overexpression of proteins was confirmed by Western blotting using antibodies against IDH2.

CRISPR-Cas9 technology was used to generate Kindlin-3 knockout cells. Two independent sgRNAs were designed by the Zhang laboratory CRISPR Design Tool. Annealed oligonucleotides were ligated into the vector LentiCRISPRv2 (Addgene, Feng Zhang’s lab) and digested with BsmBI (Fermentas). Lentiviral infection was performed with Lenti-X™ Packaging Single Shots (Clontech) in accordance with a Cleveland Clinic IBC protocol. After 72 hours, cells were selected with 2 μg/mL puromycin (Thermo Fisher Scientific) for another 48 hours. Live cells were collected and sorted by flow cytometry into 96-well plates. Individual clones were examined by western blotting or genomic DNA sequencing. The sgRNA primers sequence for K3KO are as follows: