Improved stability of GFP reporter genes on DENV2 genome with 2x 2A design.

A) Fluorescent microscopy of infected Vero cells during passaging. The left diagram outlines the passaging of a reporter virus. MOI of 0.01 was used to infect confluent Vero cells for each passage. Green-fluorescent images of infected Vero cells (right panel) were taken seven days post infection. P1, P2, P3, P4, P5, and P6 designate passage numbers. 1x 2A and 2x 2A denote the design of the reporter viruses. B) Flow-cytometry measurements of Vero cells infected with passaged DENV2-eGFP. Confluent Vero cells were infected with viruses from each passage (culture media collected at day 7) at MOI = 0.1. Infected cells were maintained for two days before whole-cell staining with 4G2 mouse antibody and Alexa-647-conjugated anti-mouse antibody. The percentage of each cell population is shown in each quardrant of the scatter plot. The top row shows the measurements of passaged DENV2-eGFP with the 1x 2A design while the bottom row show those of 2x 2A design. C) Quantification of the stability of GFP reporter genes based on flow-cytometry measurements. The stability was measured as the percentage of GFP-positive cells in the populations of infected cells (4G2-positive cells). % GFP/4G2 = (% cells with GFP)/(% cells with 4G2). D) RT-PCR of passaged DENV2-eGFP. Top diagram shows the locations of the primer binding sites (represented by arrows) on the viral genome of reporter DENV2-eGFP. Bottom is the image of agarose gel electrophoresis of the RT-PCR products of viral RNA extracted from passaged viruses.