Noninvasive Optical Imaging of Firefly Luciferase Reporter Gene Expression in Skeletal Muscles of Living Mice

Abstract

The ability to monitor reporter gene expression noninvasively offers significant advantages over current techniques such as postmortem tissue staining or enzyme activity assays. Here we demonstrate a novel method of repetitively tracking in vivo gene expression of firefly luciferase (FL) in skeletal muscles of mice using a cooled charged coupled device (CCD) camera. We first show that the cooled CCD camera provides consistent and reproducible results within +/-8% standard deviation from mean values, and a detection sensitivity (range tested: 1 x 10(4) - 1 x 10(9) plaque form-ing units (pfu)) of 1 x 10(6) pfu of E1-deleted adenovirus expressing FL driven by a cytomegalovirus promoter (Ad-CMV-FL). The duration and magnitude of adenoviral mediated (1 x 10(9) pfu) FL gene expression were then followed over time. FL gene expression in immunocompetent Swiss Webster mice peaks within the first 48 hours, falls by 98% after 20 days, and persists for >150 days. In contrast, FL activity in nude mice remains elevated for >110 days. Finally, transduced Swiss Webster and nude mice were sacrificed to show that the in vivo CCD signals correlate well with in vitro luciferase enzyme assays (r(2)=0.91 and 0.96, respectively). Our findings demonstrate the ability of the cooled CCD camera to sensitively and noninvasively track the location, magnitude, and persistence of FL gene expression. Monitoring of gene therapy studies in small animals may be aided considerably with further extensions of this technique.

In vivo imaging of mlc2v-luciferase, a cardiac-specific reporter gene expression in mice1

“…The average ex vivo signal is about 2-10-fold stronger than the in vivo signal, suggesting the absorption by overlaying tissues (eg, chest wall, skin) and blood in the ventricles. This is consistent with the observation that signal intensity from the heart was weaker than that from the superficial tissue, such as a subcutaneous tumor (17).…”

Molecular imaging of gene therapy for cancer

“…Sensitive imaging systems have been built to detect and quantitate small numbers of cells or organisms expressing luciferase as a transgene. [73][74][75] BLI has been used to monitor tumor cell growth and regression, 76,77 to visualize the kinetics of tumor cell clearance by chemotherapeutics 78 and to track gene expression. 36 Luciferase imaging has recently revealed gene transfer to tumors followed by a decrease in transgene expression with tumor cell death over time.…”

Herpesvirus saimiri-based vector biodistribution using noninvasive optical imaging

“…A region of interest (ROI) was drawn and the amount of light quantified using the maximum value of photons/s/cm 2 /steradian inside the ROI. 13 …”

“…9,10 Both firefly luciferase (which catalyses D-luciferin to produce oxyluciferin in the presence of oxygen and cofactors emitting light with a peak wavelength at 562 nm) and renilla luciferase (which catalyses the oxidation of coelenterazine producing light at 482 nm) have been used as reporter genes in living animals. [11][12][13] The major advantage of using these reporters is the minimal background fluorescence produced in animal studies, as luciferase is an insect photoprotein and not present in mammalian organisms. Bioluminescence imaging has been utilized to evaluate a number of nonviral and viral vector systems in small animal models.…”