FIG. 8.

Expression of foreign genes with JEV cDNA as the vector. (A) Schematic diagram of the pBAC^SP6/JVFLx/GFP/XbaIMBN, pBAC^SP6/JVFLx/LUC/XbaIMBN, and pBAC^SP6/JVFLx/LUC^REP−/XbaIMBN cDNA templates used for runoff transcription with SP6 RNA polymerase. Indicated are the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-driven GFP and luciferase genes that were inserted at the beginning of the 3′NTR of the viral genome, the SP6 promoter transcription start, and the runoff site generated by XbaI digestion and mung bean nuclease treatment (XbaI/MBN). In pBAC^SP6/JVFLx/LUC^REP−/XbaIMBN, a solid vertical bar indicates an 83-nucleotide deletion (nt 5581 to 5663) in the middle of the NS3 gene that determines viral translation at nt 5596 (asterisk). (B) Expression of GFP protein. BHK-21 cells were mock transfected or transfected with 2 μg of synthetic RNAs transcribed from the pBAC^SP6/JVFLx/GFP/XbaIMBN template (JVFLx/GFP/XbaIMBN), incubated for 30 h, and then fixed and examined by confocal microscopy. (C) Induction of luciferase protein. BHK-21 cells (8 × 10⁶) were mock transfected or transfected with 2 μg of synthetic RNAs transcribed from the pBAC^SP6/JVFLx/LUC/XbaIMBN (solid circles) and pBAC^SP6/JVFLx/LUC^REP−/XbaIMBN (open circles) templates and seeded in a six-well plate at a density of 6 × 10⁵ cells per well. Cells were lysed at the indicated time points, and luciferase activity was determined. The standard deviations obtained from three independent experiments are indicated by error bars.