Noninvasive Monitoring of Target Gene Expression by Imaging Reporter Gene Expression in Living Animals Using Improved Bicistronic Vectors

Plot of TK activity in N2a cells after transient transfection using 3 different plasmids (RIT, RST, and SV40-tk). For transfection experiments, 1.8 μg of each plasmid were cotransfected with 0.2 μg of pCMV-βgal into N2a cells in 6-well plates. Forty-eight hours later, cells were harvested and assayed for TK activity. Enzyme activities were normalized to β-gal activities. Error bars represent SE between triplicate samples.

Plot of TK activity in different cell lines. N2a, C6, and 293T cells were transiently transfected with plasmids RIT, RST, FIT, FST, DIT, and DST. Plasmid SV40-tk was used as a positive control. Note that TK activities are represented on a logarithmic scale (based on 100-fold of dpm/μg protein/min). Error bars represent SE between triplicate samples.

(A) Correlation of RL and TK activities from transient transfection assays in N2a cells. Cells were transfected with different concentrations of RST plasmid (2, 4, 5, 6, 8, and 10 μg). Forty-eight hours later, cells were harvested and assayed for RL and TK activities. RL and TK activities are represented as relative light units (RLU) per μg protein and dpm per μg protein per minute respectively. Error bars represent SE between triplicate observations. (B) Correlation of D2R and TK activities in transient transfection assays. Various amounts (2, 4, 5, 6, 8, and 10 μg) of CDST plasmid were transfected into N2a cells in 10-cm² dishes. Forty-eight hours later, cells were harvested and assayed for D2R and TK activities. D2R and TK activities are represented as picomoles of ³H-spiperone bound per μg of protein and dpm per μg protein per minute, respectively.

(A) Optical imaging of rl and fl reporter gene expression in vivo. Six-week old nude mice were implanted with 4 stably transfected N2a cell lines carrying RSF vector (with different levels of gene expression). Mice were imaged in CCD camera for rl expression immediately after cell implantation using coelenterazine (2 μg). Subsequent imaging was performed for fl expression using d-luciferin injected intraperitoneally (150 mg/kg). Signal intensity is represented as photons/s/cm²/sr. (B) Correlation of RL and FL activities in stably transfected N2a cells. RLU for rl are plotted against corresponding light units for fl in the 4 N2a cell lines. Expression of the 2 reporter genes shows excellent correlation (r² = 0.98).