1. Features of the BLITC reporter.
Features of the BLITC reporter. A, Schematic of the two constructs comprising the BLITC reporter. The NFAT-CBR construct (left) consists of a redshifted click beetle luciferase (CBR) followed by a polyA-signal (pA), ensuring stability and proper transport of CBR-mRNA. For activation-dependent expression, the CBR gene is under transcriptional control of the distal ARRE2 element, a consensus NFAT:AP-1 binding site of the human and murine IL2 promoter located in a 4-fold tandem upstream of a human ß-globin minimal promoter. A genomic insulator positioned downstream of the promoter site minimizes transcriptional interference from cis-acting genomic elements. The NFAT-CBR and the Rluc cassette are located on different chromosomes and inherited individually. B and C, T cells (TC) were isolated from H-Y TCR BLITC transgenic mice (MataHari TCR+ Rag1+/− NFAT-CBR+/+ Rluc+/+/CD8+) and sorted (>98%) via TCR-Vß8.3 staining and subsequently stimulated for 18 hours with anti-CD3/CD28. T cells were harvested, adjusted to cell numbers according to a dilution series, and (B) used in an in vitro luciferase detection assay or (C) injected subcutaneously into male albino RagKO mice in a 1:1 mix with matrigel, and in vivo analyzed by BLI 3 hours later, starting with NFAT-CBR. Data are representative of two independent experiments (n = 3 mice). Total flux values were determined for (B) individual wells or (C) for ROI circumscribing areas of injection. The shaded areas: respective background signals as obtained from similar ROIs placed on irrelevant mouse regions. D,In vivo images of the experiment quantified in (C). Each T-cell dose was subcutaneously injected three times across two mice and mean BLI signal values were calculated. The graphs in B and C display mean + standard error of mean (SEM).
Martin Szyska et al. Cancer Immunol Res 2018;6:
©2018 by American Association for Cancer Research