Journal: Frontiers in Oncology
Article Title: Adaptive Resistance to Immunotherapy Directed Against p53 Can be Overcome by Global Expression of Tumor-Antigens in Dendritic Cells
Figure Lengend Snippet: Retroviral transduction of D2SC/1 cells . (A) D2SC/1 cells were cultured in the supernatant of the retroviral packaging cell line FLYA4lacZ3 for 24 h and LacZ transduced cells were visualized by X-gal staining. (B) The mp53 retroviral transduction vectors are illustrated. Predicted K[d] restricted peptide presentation is marked by a box. Amino acids at position 2, 3, 5, and carboxyl termini function as anchors or auxiliary anchors within the MHC pocket. (C) Flow cytometry of mp53-transduced D2SC/1. Stable amphotropic packaging cell lines were generated by calcium phosphate transfection of FLY-AF-13 cells with pBABEpuro–p53C132F/E168G or pBABEpuro–p53M234I and puromycin selection before D2SC/1 cells were transduced three times with 1.5–3 × 10 4 CFU/ml of budding virus for 15 h. Cells were fixed and stained with 1 μg/ml PAb 421 anti-p53 antibody. Filled histograms represent stained cells transduced with the pBABEpuro control vector, open histograms D2SC/1-p53C132F/E168G, or D2SC/1-p53M234I transduced cells.
Article Snippet: The missense point mutations C132F, E168G, and M234I present in the p53 alleles were verified by sequence analysis. p53M234I and p53C132F/E168G were cloned into the retroviral transduction vector pBABEpuro (Addgene, Cambridge, MA, USA).
Techniques: Transduction, Cell Culture, Staining, Flow Cytometry, Cytometry, Generated, Transfection, Selection, Plasmid Preparation