pmaxgfp control vector  (Lonza)

Journal: Cells

Article Title: Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth

doi: 10.3390/cells9040998

Figure Lengend Snippet: PD-1 gene disruption by sgRNA/Cas9 expression vectors in EGFRvIII-targeting chimeric antigen receptor (EvCAR-T) cells derived from human peripheral T cells. ( a ) Schematic representation of the procedure for introducing PD-1 sgRNA/Cas9 expression vectors and EvCAR into human T cells. ( b ) Transfection efficacy of PD-1-targeting sgRNA/Cas9 expression vectors. The upper and lower panels depict the electroporation of control pmaxGFP vectors and PD-1-targeting sgRNA/Cas9 expression vectors in human T cells, respectively. The images were acquired under a phase contrast microscope and fluorescence microscope (left, middle), and flow cytometric data are shown on the right. ( c ) T cells at day 7 after transfection, as observed under an inverted microscope. The left, middle, and right photos show T cells, EvCAR-transduced T cells, and PD-1-targeting sgRNA/Cas9 vector- and EvCAR-transduced T cells, respectively. ( d ) Control T cells, EvCAR-transduced T cells populations and PD-1-targeting sgRNA/Cas9 vector- and EvCAR-transduced T cells populations were cultured in vitro upon stimulation with exogenous IL-2 for 21 days. The total cell number was counted on day 14 and day 21. Data show the mean ± standard deviation (SD) for four experiments. The significance of differences was determined by two-way ANOVA followed by Tukey’s test. * p < 0.05. ( e ) Representative results for the expression of PD-1 on αFab (EvCAR)-expressing T cells, as determined by flow cytometry at day 21 after electroporation. The upper and lower panels depict EvCAR-transduced T cells and PD-1-targeting sgRNA/Cas9 expression vector-transduced T cells, respectively. The left and right panels show EvCAR and PD-1 expression data, respectively. PD-1 expression was determined by gating EvCAR-positive cells. ( f ) Percentage of PD-1 positive cells (left) and normalized mean fluorescent intensity (MFI) (right) for PD-1 expression are shown. Data show the mean ± standard deviation (SD) for four experiments. The significance of differences was determined by the t-test. ** p < 0.05. ( g ) Representative data for the frequency of CD4- and CD8-positive cells among EvCAR (left) and PD-1-targeting sgRNA/Cas9 vector- and EvCAR-transduced T cell populations (right). ( h ) Frequency of CD4/CD8 double-positive, CD4/CD8 double-negative, CD4 single-positive, and CD8 single-positive cells among EvCAR and PD-1-targeting sgRNA/Cas9- and EvCAR-transduced T cells populations. Data show the mean ± standard deviation (SD) for four experiments. Significance was determined with the t-test.

Article Snippet: The PBMCs were transfected with 5 µg of the CRISPR/Cas9 expression vectors or 2.5 µg of the control pmaxGFP vector by Nucleofector 2b (Lonza, Köln, Germany), using the Amaxa Human T cell Nucleofector Kit (VPA-1002; Lonza).

Techniques: Expressing, Derivative Assay, Transfection, Electroporation, Microscopy, Fluorescence, Inverted Microscopy, Plasmid Preparation, Cell Culture, In Vitro, Standard Deviation, Flow Cytometry