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

¹ The Crump Institute for Molecular Imaging, David Geffen School of Medicine at UCLA, Los Angeles, California
² Department of Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California
³ Cedars-Sinai Medical Center, Los Angeles, California
⁴ Department of Neurobiology, Scripps Research Institute, Skaggs Institute for Chemical Biology, La Jolla, California
⁵ Department of Radiology, Bio-X Program, Stanford University School of Medicine, Stanford, California

View larger version (74K): [in a new window]   FIGURE 1. Schematic representation of imaging reporter gene expression using a SIRES-based bicistronic vector. Diagram illustrates how 2 proteins are expressed from the SIRES vector. Both gene A and gene B are coexpressed from the same vector and expression of gene B (a PET reporter) can be imaged quantitatively by trapping of a PET reporter probe (e.g., 18F-FHBG), which will be an indirect measure of expression of gene A. PCMV = CMV promoter.

View larger version (22K): [in a new window]   FIGURE 2. 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-ßgal into N2a cells in 6-well plates. Forty-eight hours later, cells were harvested and assayed for TK activity. Enzyme activities were normalized to ß-gal activities. Error bars represent SE between triplicate samples.

View larger version (25K): [in a new window]   FIGURE 3. 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.

View larger version (15K): [in a new window]   FIGURE 4. (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.

View larger version (45K): [in a new window]   FIGURE 5. (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).

View larger version (41K): [in a new window]   FIGURE 6. Optical and microPET imaging of fl and tk gene expression in vivo. For optical imaging of fl reporter gene expression, nude mice implanted with 4 different N2a cell lines (RIF, left panel; RSF, middle panel) were imaged in CCD camera immediately after cell implantation using D-luciferin (150 mg/kg). For tk gene imaging, nude mice carrying N2a tumor xenografts (RIT and RST on left and right shoulders, respectively; right panel) were imaged by microPET using 18F-FHBG (74 kBq [200 µCi]). SIRES xenograft shows significantly higher retention of reporter when compared with EMCV IRES xenograft (0.88 vs. 0.13 %ID/g tumor, respectively).