Fig. 9
From: Split intein-mediated selection of cells containing two plasmids using a single antibiotic
SiMPl allows selecting functional Jurkat T cells expressing a murine TCR on their surface. a Schematic of the SiMPl lentiviral vectors encoding TCRα and TCRβ where the puromycin acetyltransferase is split at position V82:E83. b Schematic of the workflow. c TCRα-TCRβ- Jurkat T cells selected on puromycin after transduction with the two lentiviruses in (a) are 100% positive for the TCR. Left panel, representative histograms of living cells gated using forward versus side scatter and propidium iodide staining. Numbers indicate the percentage of cells expressing murine TCRβ on their surface (TCRαβ indicates that TCRα must be expressed to allow presentation of TCRβ on the cell surface). Right panel, stacked bar chart showing the percentage of TCRαβ+ cells for the indicated conditions. Values represent mean (± standard deviation) of three independent experiments. Standard bar graphs with individual data points are shown in Supplementary Fig. 25a. d Selected cells are functional. Intracellular calcium levels after stimulation with anti-murine TCRβ antibodies for the indicated cells. Left panel, dot plots of the calcium response depicted as ratio of Fluo-3 to Fura Red fluorescence over time. Right panel, overlay of the calcium responses of puromycin selected cells transduced with 6, 12 or 24 µL of concentrated lentivirus. The graph shows the 70th percentile of the ratio of Fluo-3 to Fura Red fluorescence over time. Source data are provided as a Source Data file
