Source: Sterner, R. M. et al., Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells. J. Vis. Exp. (2019)
The video outlines a process for creating genetically modified CAR T cells through the CRISPR-Cas9 System. Infecting T cells with CRISPR and CAR lentiviruses results in modifications to the target gene and the synthesis of a chimeric antigen receptor or CAR, ultimately leading to the formation of genetically modified CAR T cells.
1. CART19 cell production
2. Granulocyte macrophage colony-stimulating factor (GM-CSF k/o) CART19 production
The authors have nothing to disclose.
CD3 Monoclonal Antibody (OKT3), PE, eBioscience | Invitrogen | 12-0037-42 | |
CD3 Monoclonal Antibody (UCHT1), APC, eBioscience | Invitrogen | 17-0038-42 | |
Choice Taq Blue Mastermix | Denville Scientific | C775Y51 | |
CTS (Cell Therapy Systems) Dynabeads CD3/CD28 | Gibco | 40203D | |
CytoFLEX System B4-R2-V2 | Beckman Coulter | C10343 | Flow cytometer |
Dimethyl sulfoxide | Millipore Sigma | D2650-100ML | |
Dulbecco's Phosphate-Buffered Saline | Gibco | 14190-144 | |
Dynabeads MPC-S (Magnetic Particle Concentrator) | Applied Biosystems | A13346 | |
Easy 50 EasySep Magnet | STEMCELL Technologies | 18002 | |
EasySep Human T Cell Isolation Kit | STEMCELL Technologies | 17951 | Negative selection magnetic beads; 17951RF includes tips and buffer |
Fetal bovine serum | Millipore Sigma | F8067 | |
FITC Mouse Anti-Human CD107a | BD Pharmingen | 555800 | |
Fixation Medium (Medium A) | Invitrogen | GAS001S100 | |
GenCRISPR gRNA Construct: Name: CSF2 | GenScript | N/A | Custom order |
CRISPR guide RNA 1; Species: Human, Vector: | |||
pLentiCRISPR v2; Resistance: Ampicillin; Copy number: | |||
High; Plasmid preparation: Standard delivery: 4 μg (Free | |||
of charge) | |||
Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 | Invitrogen | A-21235 | |
https://tide.nki.nl. | Desktop Genetics | ||
Human AB Serum; Male Donors; type AB; US | Corning | 35-060-CI | |
IFN gamma Monoclonal Antibody (4S.B3), APC-eFluor 780, eBioscience | Invitrogen | 47-7319-42 | |
Lipofectamine 3000 Transfection Reagent | Invitrogen | L3000075 | |
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation | Invitrogen | L34966 | |
Lymphoprep | STEMCELL Technologies | 7851 | |
Monensin Solution, 1000X | BioLegend | 420701 | |
Mouse Anti-Human CD28 Clone CD28.2 | BD Pharmingen | 559770 | |
Mouse Anti-Human CD49d Clone 9F10 | BD Pharmingen | 561892 | |
Mouse Anti-Human MIP-1β PE-Cy7 | BD Pharmingen | 560687 | |
Mr. Frosty Freezing Container | Thermo Scientific | 5100-0001 | |
NALM6, clone G5 | ATCC | CRL-3273 | Acute lymphoblastic leukemia cell line |
Nuclease Free Water | Promega | P119C | |
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.22uM, sterile | Genesee Scientific | 25-227 | |
Olympus Vacuum Filter Systems, 500 mL, PES Membrane, 0.45uM, sterile | Genesee Scientific | 25-228 | |
Opti-MEM I Reduced-Serum Medium (1X), Liquid | Gibco | 31985-070 | |
PE-CF594 Mouse Anti-Human IL-2 | BD Horizon | 562384 | |
Penicillin-Streptomycin-Glutamine (100X), Liquid | Gibco | 10378-016 | |
Permeabilization Medium (Medium B) | Invitrogen | GAS002S100 | |
PureLink Genomic DNA Mini Kit | Invitrogen | K182001 | |
Puromycin Dihydrochloride | MP Biomedicals, Inc. | 210055210 | |
QIAquick Gel Extraction Kit | QIAGEN | 28704 | |
Rat Anti-Human GM-CSF BV421 | BD Horizon | 562930 | |
RoboSep-S | STEMCELL Technologies | 21000 | Fully Automated Cell Separator |
SepMate-50 (IVD) | STEMCELL Technologies | 85450 | |
Sodium Azide, 5% (w/v) | Ricca Chemical | 7144.8-16 | |
X-VIVO 15 Serum-free Hematopoietic Cell Medium | Lonza | 04-418Q |
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