Noninvasive Optical Imaging of Firefly Luciferase Reporter Gene Expression in Skeletal Muscles of Living Mice  Joseph C. Wu, Gobalakrishnan Sundaresan, Meera Iyer, Sanjiv S. Gambhir  Molecular Therapy  Volume 4, Issue 4, Pages 297-306 (October 2001) DOI: 10.1006/mthe.2001.0460 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 The Xenogen In Vivo Imaging System (IVIS) consists of a cooled CCD camera mounted on a light-tight imaging chamber, a cryogenic refrigeration unit, a camera controller, and a computer system for data analysis. The “calibration standards” show a picture of a hockey puck drilled with four holes and covered with glass (inset). Each hole contains carbon-14 isotopes (T1/2 = 5730 years) of various concentrations (100 μCl, 10 μα, 100 nCi, and 10 nCi) immersed in scintillation cocktail. Light emitted from β-decay is detected by the cooled CCD camera, converted into photons, and transformed to relative light units (RLU) on images. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Light activities emitted from carbon-14 isotopes were followed over 4 months before in vivo animal imaging. These fixed light sources serve as calibration standards for measuring the reproducibility of the IVIS. (A) For 10 μCl isotope, the maximum RLU obtained by the cooled CCD camera using six different image acquisition times (10–60 s) are shown for individual time points. (B) The average of all maximum CCD RLU for each acquisition time is plotted for both 10 μCl isotope (bold line; left y axis) and 1 μCl isotope (dashed line; right y axis). Error bars represent S.D. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 The kinetics of FL activity were followed immediately after intraperitoneal injection of substrate luciferin (15 mg/ml solution; 126 mg/kg body weight) with repetitive imaging by the cooled CCD camera. The peak FL activity was observed within 15–25 min after luciferin injection. The five highest values were averaged to represent the magnitude of FL activity for that particular day. FL activity was still detected up to 2–3 h later. High FL activity is observed at day 2 (A), followed by significant reductions at day 6 (B) and day 20 (C) (both P < 0.05). Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Detection sensitivity of the cooled CCD camera is affected by the host immune system and the presence of white fur and tissue layer. (A) Serially diluted titers of Ad-CMV-FL (1 × 109, 1 × 108, 1 × 107, 1 × 106, 1 × 105 and 1 × 104 pfu) were injected into thigh skeletal muscles of Swiss Webster (dotted box) and nude mice (shaded box). At day 6, high FL activity was seen in nude mice using high viral titer (1 × 109) and a lower activity in Swiss Webster mice. RLU/min decreases with successive lower viral titer for both mice strains. The lower limit of detection for Swiss Webster mice is 1 × 106 pfu (244 ± 33 RLU/min). The lower limit of detection for nude mice is 1 × 104 pfu (739 ± 472 RLU/min). Control virus yielded only background signals for both strains (53 ±11 RLU/min). (B) Swiss Webster mice (n = 4) were imaged intact (dotted box), shaved (striped box), and skinned (dark box) 2 days after injection of 1 × 109 pfu Ad-CMV-FL into thigh skeletal muscles. There is an 18% increase of RLU/min with furs shaved and an additional 33% with skin tissue layer exposed (*P < 0.05 both). Removing the skin tissue layer for nude mice increased RLU/min by 19%, but the difference is not statistically significant (P = 0.11). Error bars represent S.E.M. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 The magnitude and duration of FL gene expression in different host immune systems can be monitored noninvasively and repetitively using the cooled CCD camera. The y axis shows the maximum CCD RLU/min in log scale and the × axis shows the number of days. (A) For Swiss Webster mice, the highest FL activity occurred at day 2 and was significantly reduced with following days (P < 0.01 at days 4, 6, 8, and so forth). (B) For nude mice, a considerable amount of FL activity was seen throughout the study period. Nevertheless, there is a definite trend for gradual peaking as well as gradual reduction of FL activity over time. (C, D) A composite of cooled CCD images on the same animal for Swiss Webster and nude mice, respectively. FL activity was also seen in the liver of nude mice starting at day 20 and earlier for other nude mice. Control virus yielded only background signals for both strains (55 ± 10 RLU/min). Error bars represent S.E.M. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Lower titers of Ad-CMV-FL followed over time in nude mice. All titers showed delayed peaking followed by gradual reduction of FL activity. For 1 × 104 pfu, FL activity was detected starting at day 6 and disappeared by day 115. Error bars represent S.E.M. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 Correlation of FL activity from in vivo CCD images (RLU/min) versus standard in vitro luminometer assays (RLU/mg protein) are shown on x and y axes, respectively. (A) For Swiss Webster mice, r2 = 0.91 and the slope of regression line is 38.9 (n = 34 muscle groups). (B) For nude mice, r2 = 0.96 and slope of regression line is 28.5 (n = 24 muscle groups). Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 8 The magnitude and duration of FL gene expression can also be followed in other organs or tissues. For hepatic studies, mice were injected with 1 × 109 pfu Ad-CMV-FL via the tail vein. Most of the virus localized to hepatocytes due to the presence of coxsackie-adenovirus receptors. Peak FL activity was observed between days 2 and 6. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 9 The effect of humoral immunity against repeated adenovirus injection can be studied using the cooled CCD camera. Mice injected with 1 × 109 pfu of Ad-CMV-FL into right thigh showed considerable FL activity at day 2. The same viral titer was injected into contralateral thigh after 20 days (second) and 40 days (third injection). At day 2, FL activity from left thigh was significantly suppressed after the second administration (262 ± 212 RLU/min; *P < 0.01) and barely detectable following the third administration (94 ± 7 RLU/min; *P < 0.01). The background noise level is 56 ± 3 RLU/min. The error bar represents S.E.M. Molecular Therapy 2001 4, 297-306DOI: (10.1006/mthe.2001.0460) Copyright © 2001 American Society for Gene Therapy Terms and Conditions