The EGFRvIII-specific CAR vector has been previously described (12 (link),34 (link)). Briefly, second generation CARs were defined by the intracellular signaling components CD28 and CD3z, and third generation CARs were defined by CD28, 4–1BB, and CD3z. To produce CARs deficient in Lck signaling, two amino acid substitutions were introduced in the PYAP motif in the CD28 signaling domain of the CAR transgene through site-directed mutagenesis (30 (link)). CARs were generated by retroviral transduction (35 (link)). Briefly, retroviral supernatant was produced by cotransfection of HEK 293T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen) with MSGV1-EGFRvIII CAR retroviral vectors and pCL-Eco helper plasmid (Imgenex). On the same day, splenocytes were freshly harvested from C57BL/6NCr mice (Charles River Laboratories) and cultured in R10 mouse T-cell media supplemented with 50units/mL IL-2 and 2.5μg/mL Concanavalin A. After 48 hours, splenic T cells were transduced with retroviral supernatant on non-tissue culture 24-well plates previously coated with 0.5 mL of RetroNectin (Clontech) at a concentration of 25 μg/mL in PBS. Cells were plated at a density of 1×106/mL in viral supernatant supplemented with 50units/mL IL-2. Cells were split every 24 hours for two days.
Pcl eco helper plasmid
Manufactured by Novus Biologicals
The PCL-Eco helper plasmid is a DNA construct used in molecular biology applications. It contains genetic elements necessary for the maintenance and replication of the plasmid in bacterial host cells.
Automatically generated - may contain errors
Lab products found in correlation
Retronectin, by Takara Bio (2 mentions)
C57bl 6ncr mice, by Charles River Laboratories (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Mscv ltrmir30 pig lmp vector, by Thermo Fisher Scientific (1 mentions)
Platinum e plat e retroviral packaging cell line, by Cell Biolabs (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
T blunt vector, by Solgent (1 mentions)
3 protocols using pcl eco helper plasmid
1
Generation of EGFRvIII-specific CAR T cells
2
Cloning and Knockdown of Mouse Etv5 and Maf
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The amino-terminal FLAG-tagged coding sequence (CDS) of mouse Etv5 was amplified by PCR, initially cloned into T-blunt vector (SolGent), and then verified by sequencing. A confirmed Etv5 CDS fragment digested by XhoI/HpaI was sub-cloned into MigR1 retroviral vector (MigR1-ETV5-GFP). The shRNA expression vectors for knockdown of mouse Etv5 and Maf were generated using MSCV-LTRmiR30-PIG (LMP) vector (Open Biosystems) according to the manufacturer’s instruction. The target sequences were as follows. For shETV5: 5′-ACCCGAGAGACTGGAAGGCAAA-3′ and for shMAF: 5′-AAGATATAACCTGCAAGCATAT-3′.
Viruses were generated by transient co-transfection of Platinum-E (Plat-E) retroviral packaging cell line (Cell Biolabs) with the cloned retroviral vectors and pCL-Eco helper plasmid (Imgenex). Briefly, 0.5–0.8 × 106 plat-E cells were plated in 6-well plates. On the next day, the cells were transfected with 1.2 μg of retroviral vector and 0.8 μg of pCL-Eco using FuGENE HD transfection reagent (E2311, Promega). Retrovirus-containing supernatants were collected 48 h later and frozen at −80 °C.
Viruses were generated by transient co-transfection of Platinum-E (Plat-E) retroviral packaging cell line (Cell Biolabs) with the cloned retroviral vectors and pCL-Eco helper plasmid (Imgenex). Briefly, 0.5–0.8 × 106 plat-E cells were plated in 6-well plates. On the next day, the cells were transfected with 1.2 μg of retroviral vector and 0.8 μg of pCL-Eco using FuGENE HD transfection reagent (E2311, Promega). Retrovirus-containing supernatants were collected 48 h later and frozen at −80 °C.
3
Generation of EGFRvIII-Targeted CAR T Cells
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The EGFRvIII-specific CAR is a third generation vector defined by inclusion of the CD28, 4-1BB, and CD3ζ intracellular signaling moieties on the CAR transgene. The single-chain variable fragment is derived from human monoclonal antibody (mAb) 139 and has been previously described.9 (link) To produce CARs, retroviral supernatant was produced by co-transfection of HEK 293 T cells using Lipofectamine 2000 Transfection Reagent (Invitrogen), the CAR retrovirus, and pCL-Eco helper plasmid (Imgenex). On the same day as transfection, fresh spleens were isolated from donor C57 BL/6NCr mice (Charles River Laboratories) and manually disrupted on a 10 cm dish using a plunger. Red blood cells (RBCs) were lysed, and splenocytes were cultured in mouse T-cell media supplemented with 50 U/mL IL-2 and 2.5 µg/mL Concanavalin A. After 48 hours, splenic T cells were transduced with retroviral supernatant (CAR alone or 1:1 with CAR and firefly luciferase) on non-tissue-culture 24-well plates previously coated with 0.5 mL of RetroNectin (Clontech) at a concentration of 25 µg/mL in PBS. Cells were plated at a density of 1 × 106/mL in viral supernatant supplemented with 50 U/mL IL-2. Cells were split every 24 hours for two days.
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