Figure 2

Generation and characterization of the reporter MCMV MIEP^r. (A) Graphic representation of the MIEP^r reporter construct and its integration into the MCMV genome. The bidirectional major immediate early promoter enhancer (MIEP) was flanked by the yellow fluorescent protein (YFP) and tdTomato (Tom) and inserted ectopically into the MCMV genome, replacing the viral genomic region between the genes m07 and m17. (B) LSEC-uniLT were infected with 1 MOI of MIEP^r and representative pictures were visualized by epifluorescence microscopy at indicated hours post infection (hpi) are shown. (C) NIH-3 T3 cells were infected with MIEP^r or MCMV wild type (WT) at an MOI of 0.1 and virus growth was monitored by plaque assay of cell supernatants at indicated days post infection (dpi). Averages (+/- SD) from three independent experiments are shown. The dashed line represents the limit of detection (DL). (D) Endogenous (ie1 and ie2) and reporter (YFP and Tom) transcripts were measured by qRT-PCR. The cDNA was synthesized from RNA obtained from LSEC-uniLT infected at an MOI of 10 for the indicated hours post infection (hpi). Copy numbers were normalized to GAPDH and are represented as averages (+/- SD) from three independent experiments.