Figure 2

Artificial reporter vectors with and without a Leader sequence. (a) Diagram for the vector design. Leader sequence (labeled as Leader-S in the box) from SARS-CoV was synthesized and inserted upstream of a GFP or RFP reporter gene driven by an EF-1α or CMV promoter as indicated. a–e are primers described in Materials and methods. (b) RT-PCR results showing the mRNAs from the reporter vectors. 293T cells were transfected with pEFBos/L-GFP (lanes 1 and 5), pEFBos/GFP (lanes 2 and 6), pDsRed1.1/RFP (lanes 3 and 7), pDsRed1.1/L-RFP (lanes 4 and 8). The total RNA was isolated for performance of RT-PCR using the primers as indicated. The predicted bands are listed above the gel. The upper bands (noted as Leader-S+Reporter) are the mRNA from reporter constructs with the Leader sequence of SARS-CoV and the lower bands (noted as Reporter) are from reporter constructs without the Leader sequence. DNA ladder is shown on the left of the gel. The PCR reaction condition was 25 cycles of denaturation (94°C, 30 s), annealing (50°C, 1 min) and extension (72°C, 1 min).