Imaging Transgene Expression in Live Animals Alik Honigman, Evelyn Zeira, Patricia Ohana, Rinat Abramovitz, Einat Tavor, Iris Bar, Yoram Zilberman, Rosalia Rabinovsky, Dan Gazit, Aviva Joseph, Amos Panet, Ela Shai, Aaron Palmon, Morris Laster, Eithan Galun  Molecular Therapy  Volume 4, Issue 3, Pages 239-249 (September 2001) DOI: 10.1006/mthe.2001.0437 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Kinetics of light emission following injection of luciferin. (A) Two days following IV injection of 107 AdSV40/luc, mice were anesthetized and luciferin was injected IP. Pictures were taken at the indicated time intervals. (B) Thirty days after subcutaneous implantation of T50/Luc in C3H mice, the mice were anesthetized and light emission was monitored at the indicated time intervals following IP injection of luciferin. (C) pLNC/Luc naked DNA (10 μg) was electroporated following injection into the leg muscle of BALB/c mice. At day 23 following the electroporation, luciferin was IP injected and the CCCD camera monitored light emission at the indicated time intervals. Light measurements are expressed as integrated light units. In all the experiments shown the exposure and acquisition time was 2 min. The curves represent the average of integrated light units at each time point measured in each experiment. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Kinetics of light emission following injection of luciferin. (A) Two days following IV injection of 107 AdSV40/luc, mice were anesthetized and luciferin was injected IP. Pictures were taken at the indicated time intervals. (B) Thirty days after subcutaneous implantation of T50/Luc in C3H mice, the mice were anesthetized and light emission was monitored at the indicated time intervals following IP injection of luciferin. (C) pLNC/Luc naked DNA (10 μg) was electroporated following injection into the leg muscle of BALB/c mice. At day 23 following the electroporation, luciferin was IP injected and the CCCD camera monitored light emission at the indicated time intervals. Light measurements are expressed as integrated light units. In all the experiments shown the exposure and acquisition time was 2 min. The curves represent the average of integrated light units at each time point measured in each experiment. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Localization and intensity of light emitted from subcutaneous implanted tumor cells. (A) T50/Luc mouse bladder cells (2 × 105) were subcutaneously implanted in C3H mice. Pictures were taken between day 4 and day 28 following implantation (A–C). The tumors were excised at day 28, photographed (D), and exposed to the CCCD camera for monitoring Luc-activated light (E) in petri dishes. HepG2/Luc cells (1.5 × 107) were administrated SC into Balb/C nude mice. Pictures were taken at various time intervals. The image of the tumor at day 40 is shown (F). (This picture was taken by the Hamamatsu Camera, exposure time 20 minutes.) (G) Correlation between tumor growth as defined by the camera in pixels and emission of light expressed as integrated light/2-min exposure and acquisition, along the time scale (in days). The values indicated on the wedge color scale are the maximum and the minimum of the gray level. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Localization and intensity of light emitted from subcutaneous implanted tumor cells. (A) T50/Luc mouse bladder cells (2 × 105) were subcutaneously implanted in C3H mice. Pictures were taken between day 4 and day 28 following implantation (A–C). The tumors were excised at day 28, photographed (D), and exposed to the CCCD camera for monitoring Luc-activated light (E) in petri dishes. HepG2/Luc cells (1.5 × 107) were administrated SC into Balb/C nude mice. Pictures were taken at various time intervals. The image of the tumor at day 40 is shown (F). (This picture was taken by the Hamamatsu Camera, exposure time 20 minutes.) (G) Correlation between tumor growth as defined by the camera in pixels and emission of light expressed as integrated light/2-min exposure and acquisition, along the time scale (in days). The values indicated on the wedge color scale are the maximum and the minimum of the gray level. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Implantation of Luc-expressing cells. (A) HepG2/Luc cells (2.5 × 106) were injected into the spleen and light was monitored at various time points thereafter. Light emitting from the liver at day 40 following the injection to the spleen is presented. (This picture was taken by the Hamamatsu Camera, exposure time 20 min.) Human prostate cells (2 × 106), PC3.38/Luc, were implanted orthotopically in the prostate of a SCID mice and exposed to the CCCD camera for 2 min at day 30 (B). Development of metastasis (“M”) on day 44 (C) can be seen. T50/Luc cells (2 × 106) were introduced by cannulation into the tibia of a Sand rat and pictures were taken at different days. Growth of the tumor can be seen at day 21 (D) to day 28 (E). The tumor was excised and placed in a petri dish under the camera. The bone (“B”) and the tumor (“T”) can be seen in (F) and light detected ex vivo from the same tumor tissue can be seen in (G). Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Expression of luc in the liver, following infection with AdSV40/Luc. Recombinant AdGL3Luc viral particles (107) carrying the luc under the control of the constitutive SV40 promoter were administrated IV to BALB/c mice. Light emission was monitored at time intervals as indicated (A–D). The liver of mice was excised at days 6 and 10 following IV injection. The liver was exposed to the CCCD camera immediately in light (E, G) and light emitting from the liver was monitored for 2 min (F, H). Quantification results expressing the kinetics of Luc activity as monitored by the CCCD camera in arbitrary light units are shown (I). Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Direct injection of AdSV40/luc to organs. The recombinant AdSV40/Luc particles (50 μl containing 108) were delivered by retrograde injection to the mouse salivary gland (A and B); into the bladder of rats by a catheter (C); and injected to the leg muscle of a mouse (E). Luc expression was detected in the salivary gland 24 h (A) and 30 d (B) after infection; in the bladder at 45 h (C); and in the muscle 6 d following injection (E). The bladder was removed from the rat and most of the light-producing cells were removed (D). Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Luc expression from the leg muscle of mice following injection and electroporation of naked pLNC/Luc DNA. (A–D) We injected 3, 5, 10, and 15 μg, respectively, of pLNC/Luc into the leg muscle of BALB/c mice, followed by electroporation. (E–H) Time kinetics of luc expression following injection and electroporation of 15 μg of pLNC/Luc. The contralateral leg is always injected with the same dose of DNA without electroporation. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 Luc expression following transfection with naked plasmid DNA. (A) Transfection into the bladder of a rat with bladder carcinoma induced by the carcinogen BBN. CaPO4 precipitate of pH19/Luc (100 μg) was introduced by a catheter into the bladder of a rat pretreated with BBN (15 weeks). The luc gene under the control of the H19 promoter is expressed after 32 h (B). pLNC/Luc plasmid DNA (100 μg) was introduced IV by the high-velocity method. Light was monitored on day 30 (C). Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 8 Imaging of mice injected with recombinant adenoviruses expressing luc under the control of the HIV-1 LTR constitutive promoter. Recombinant viral particles (1011) carrying luc under the control of the constitutive HIV-1 LTR promoter were administrated to BALB/c mice either IV (A) or IP (B). Pictures taken at day 9 are presented. Light is detected in the region above the testis. Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 9 Transgenic mice expressing luc under the control of a liver-specific promoter, C/EBPβ (Plap), and through a conditional expression system activating the specific liver promoter at the absence of tetracycline. (A) Transgenic mice expressing luc under the control of the human osteocalcin (BGLAP) promoter. The luc activity was detected at the site of long bone formation and teeth development (B and C). Molecular Therapy 2001 4, 239-249DOI: (10.1006/mthe.2001.0437) Copyright © 2001 American Society for Gene Therapy Terms and Conditions