Noninvasively observing processes within a living organism has long been a goal for many researchers. As early as the 1940s researchers were creating surgical windows to view events within organisms. Vital dyes have also been used to track events and cellular movements within an organism. Researchers are now adapting reporter assays, and even luminescent enzyme assays, traditionally used with cultured cells or extracts, to gain temporal and spatial information about biological processes within an entire organism(1)
The current availability of luciferase substrates that produce light upon activation (e.g., VivoGlo™ Caspase-3/7 Substrate; Figure 1) enable spatio-temporal measurement of multiple molecular events and pathways (apoptosis, drug metabolism, proteasome degradation and others) in living mice.
Ideally a reporter system for in vivo imaging would allow a researcher to follow a particular cellular or molecular process over an extended time frame, would be sensitive enough to reveal physiological changes over time, and would not interfere with naturally occurring processes, for instance due to the overexpression of a protein. The imaging technology also would need to produce signal with very low background and be able to detect an optical signal that passes through the tissues of a living organism.
Researchers have demonstrated repeatedly that luciferase-based assays are exquisitely sensitive. Since the luciferase gene can be stably transfected into cells under the control of almost any promoter, signal will not be diluted following cell division as with the signal from a vital dye. Additionally, luciferase assays in mammalian systems are are not subject to high background as a result of tissue autofluorescence(1)
. This is a distinct advantage of luminescence over fluorescence.
In this paper we describe a line of reporter mice (repTOP™ mice; Figure 1) engineered for in vivo imaging studies based on the ubiquitous expression of the luciferase reporter gene(6)
. The current availability of luciferase substrates that produce light upon activation (e.g., VivoGlo™ Caspase-3/7 Substrate; Figure 1) enable spatio-temporal measurement of multiple molecular events and pathways (apoptosis, drug metabolism, proteasome degradation and others) in living mice. Here we demonstrate the use of VivoGlo™ technology to measure liver apoptosis by bioluminescence imaging in repTOP™ mice.
Materials and Methods
The repTOP™; technology allows repTOP™ mice to express the luciferase reporter gene ubiquitously. The technology is based on the use of Matrix Attachment Regions (MAR) in transgenesis (Patent EP 1913813A1), which shield the expression of the reporter cassette from the influence of the surrounding chromatin (6)
and on the appropriate integration of the transgene into a constitutively open locus in the mouse genome.
VivoGlo™ Caspase-3/7 Substrate
VivoGlo™ Caspase-3/7 Substrate is a firefly luciferase substrate protected by the caspase-3 and -7 peptide sequence. Upon activation of caspase-3 or -7, the DEVD-peptide is cleaved to release aminoluciferin. When used in a suitable animal model containing firefly luciferase, the liberated aminoluciferin will react with luciferase to generate measurable light.
Measurement of Induced Liver Apoptosis in Tissue Extract
Mice were treated with 800 mg/kg d-GalN (Sigma) + 100μg/kg LPS (Sigma)(9)
or vehicle (PBS) only for six hours. Protein extracts were prepared from the livers of treated and control mice. Caspase-3/7 activity was assessed using immunoblots with anti-cleaved caspase (Asp175) and antiprocaspase polyclonal antibodies (Cell Signaling) and a luminescent caspase-3/7 activity assay (Caspase-Glo® 3/7 Assay).
In Vivo Imaging of Caspase-3/7 Activity in Mouse Liver
Liver apoptosis was induced in repTOP™ male mice with a single intraperitoneal injection of vehicle or
d-GalN + LPS (treated; 4 mice/experimental group). Six hours after treatment, mice were given intraperitoneal injections with three different doses of VivoGlo™ Caspase-3/7 Substrate (17, 50 and 150mg/kg) and then subjected to a 35-minute time course analysis carried out with a sequence of in vivo imaging sessions (5 minutes each). Photon emission from different body areas of the treated mice was quantified (photons/s/cm2/sr) by a CCD-camera (Caliper Life Sciences) and normalized over emission from mice treated with vehicle.
Ex Vivo Imaging of Caspase-3/7 Activity
Ex vivo analysis was carried out in liver, white adipose tissue (WAT), intestine, kidney and heart tissues dissected from repTOP™ mice euthanized after the in vivo imaging session from both vehicle (V) and d-GalN + LPS-treated (T) mice.
Results and Discussion
Treatment of repTOP™ mice with 800mg/kg d-GalN + 100μg/kg LPS clearly induced caspase-3/7 activity as assessed by immunoblot and caspase activity assay (Figure 2).
A dose-dependent increase in photon emission was detectable in the hepatic area with a maximal cleavage of DEVD-peptide by caspase-3/7 between 15 and 30 minutes after substrate injection (Figure 3, Panel A). No comparable induction was seen in the remaining body regions analyzed, in line with the apoptotic treatment adopted. Data represent the average of four measurements. As a control, we tested whether the apoptotic treatment influenced luciferase expression in the liver of repTOP™ mice by treating mice with unmodified luciferin, directly indicating the levels of luciferase expression (Figure 3, Panel B). Bioluminescence imaging data clearly demonstrate that there were no differences in photon emission between vehicle or d-GalN + LPS (treated) groups. Bioluminescence was below levels of detection in mice not injected with the substrate (n.d., not detectable) but was in the range of linearity of the system in mice injected with 75 mg/kg luciferin. Data represent the average of four measurements +/– sem.
To confirm and extend in vivo imaging data, ex vivo analysis was carried out in liver, white adipose tissue (WAT), intestine, kidney and heart tissues dissected from repTOP™ mice euthanized after the in vivo imaging session from both vehicle (V) and d-GalN + LPS-treated (T) mice (Figure 4). Quantification of photon emission in each single organ proves a clear induction of caspase-3/7 activity selectively in liver of treated mice. Thus, the dose-dependent increase in photon emission previously highlighted in the hepatic area by the in vivo analysis is fully confirmed in dissected liver. In addition, the ex vivo imaging highlights caspase-3/7 activation in WAT. In the mouse model of hepatic failure, no apoptosis in the adipose tissue was previously reported, and this demonstrates the power of the new methodology to discover unexpected sites of apoptosis.
This paper presents the first evidence of the possibility to combine reporter mice and modified luciferase substrates that are activated by an enzymatic reaction for in vivo imaging of molecular events. The technological synergy of the VivoGlo™ reagents and repTOP™ mice will open new avenues to study multiple molecular pathways in a single complex living organism.
repTop™ Mice are available as frozen embryos or breading pairs at TOP SRL. For more information and purchasing contact: TOP SRL.