|Home | About | Journals | Submit | Contact Us | Français|
During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in its infancy, reporter animals already represent a valuable tool for biomedical investigation. The present chapter aims at critically illustrating the methodology to be applied when dealing with reporter systems and in vivo imaging.
Intracellular receptors (IR) belong to a large family of molecules of growing interest because of their relevance for the control of reproductive and major metabolic functions in mammals (1). Most IR are present in a wide variety of cells and tissues and some of them may be considered as ubiquitously expressed. IR are transcription factors regulated by lipophylic hormones. Upon binding to their cognate ligands IR undergo a series of structural modifications facilitating the attraction of co-regulators thus inducing/blocking the formation of the transcription initiation complex (2, 3). Co-regulators are indispensable elements for IR activity; because of that, IR action may be finely tuned depending on the specific tridimensional conformation induced by the given ligand and by the accessibility of co-activators and co-repressors. The availability of co-activators and co-repressors is cell-specific and variable in time (4, 5). This “tripartite” mechanism of action provides IR with a significant variety of effects on transcription depending on the cell and promoter taken in consideration. The peculiar mechanism of action and the wide distribution of IR has represented an obstacle for the identification of their precise physiological functions and for the generation of drugs with a well-focused therapeutic activity.
However, the nature of transcription factors has facilitated the generation of novel experimental model systems, like reporter mice. Applying molecular imaging with reporter mice enables one to investigate IR spatio-temporal activity in living organisms. Such an approach facilitates the understanding of their pathophysiological activity and the identification of drugs devoid of major side-effects (6, 7). These animals have already shown their potential for physiological and pharmaco-toxicological studies (8–10). The present protocol aims at indicating the modalities for the application of reporter mice engineered to express a bioluminescent protein in biological studies as exemplified by showing data obtained in our lab with the ERE-Luc reporter mouse model.
With the term reporter mice we refer to animals genetically engineered to express a reporter gene under the control of IR responsive promoters. Appropriate care must be taken to ensure that the reporter is not susceptible to local restraint (such as silencers or enhancers) and that, in all cells, is freely accessible to the activated IR and to the transcriptional apparatus. From a theoretical point of view, several protocols may be applied to the generation of reporter animals; however, to our knowledge, only a few of the models currently available ensure an expression of the reporter which is ubiquitous and appropriately reflects the state of activity of the receptor (see Notes 1, 2).
The strategy followed by our laboratory in the production of transgenic reporter mice consists in the exploitation of insulator sequences. For the generation of the transgenic reporter mouse, the transgene to be injected in the male pronucleus is flanked by insulator sequences (such as matrix attachment regions –MAR- and beta-globin hypersensitive site 4 –HS4-): this has enabled the production of animals where the expression of the reporter is ubiquitous and regulated by the hormone-receptor complex (11–13).
The promoter driving the reporter transcription is generally constituted by a sequence from the TK promoter ensuring a basal level of transcription and by a multimerized hormone responsive element (HRE): the exact location of the multimerized HRE and the number of its units is selected experimentally with the creation of a series of stably transfected cell lines where the expression of the reporter in the absence/presence of hormonal stimulation is evaluated (9, 11).
The reporter is selected among genes encoding for proteins with a short turn-over rate (to enable the dynamic view of the state of promoter activity) and easily detectable by non-invasive imaging technologies. So far, we have generated mainly reporter animals for bioluminescence employing as a reporter the fire-fly luciferase: this choice was driven by the short half life of the enzyme and by the relatively easy and cost efficient application of bioluminescence imaging (BLI) to small animals (14).
In mice carrying as a reporter luciferase, the activity of the promoter may be evaluated: (a) directly in living animals by measuring photon emission resulting from luciferase enzymatic activity; (b) ex vivo in tissues dissected from the animal; (c) by measuring luciferase enzymatic activity in tissue extracts.
The possibility to measure photon emission by means of CCD cameras has the advantage that the effect of a given compound or physiological changes can be measured by observing a single animal over time. The disadvantage of this approach is that the equipment available at present time provides bi-dimensional analysis of photon emission and does not allow the investigator to acquire an unequivocal estimate of the exact origin of photon emission. Furthermore, it is well known that the different layers of tissue shield the photons, therefore impairing the measurements of luciferase activity from the inner most organs. Thus, the in vivo analysis must be complemented either by ex vivo imaging of the tissues immediately after isolation from the reporter animal or by the quantitative measurement of the enzymatic activity of luciferase accumulated in the tissues of interest.
A detailed description of the generation of a reporter mouse was previously described by Ciana et al. (11). The different steps can be summarized as follows:
In vivo imaging requires the use of specific CCD slow-scan cameras equipped with appropriate image processors and software for image reconstruction and localization of photon emission. For the bioluminescence detection:
Preliminary experiments are needed aimed to ensure that the substrate luciferin reaches all tissues at saturating concentrations, these studies are carried out first injecting the mice with a high dose of luciferin (e.g., 150 mg/kg) and measuring photon emission at for 5 min sessions at 5, 10, 15, 20, 25, 30, 35, 40 min after substrate injection. Photon emission is calculated in head, limbs, thymus, chest, abdomen, limb, and tail. This experiment will enable the investigator to evaluate the time necessary for the substrate to reach all parts of the body (typically between 5 and 40 min). The time selected will be used for a second experiment aimed at establishing the dosage of luciferin necessary and sufficient to saturate the enzyme in all body regions. This second experiment will be done by injecting the animals with increasing concentrations of luciferin (typically, 8–80 mg/kg, i.p.) and exposing them to the CCD camera for a 5 min session at the time selected in the previous experiment (Fig. 20.2). The concentration of luciferin giving maximal response in all organs is then selected for future experiments.
Ex vivo bioluminescence imaging assay is simply carried out by exposing the dissected organs to the CCD camera. To this aim, animals need to be injected with luciferin as before. Generally, once the imaging session ends, the tissue is rapidly frozen for the preparation of tissue extracts where luciferase enzymatic activity can be measured.
Ex vivo imaging is of particular interest for the study of luciferase activity in the central nervous system. In this case, due to the limited diffusion of luciferin through the blood brain barrier, the substrate is administered by intracerebroventricular (ICV) injection in anesthetized animals. The injection is done according to specific stereotaxic coordinates (Bregma, −0.25 mm; lateral, 1 mm; depth, 2.25 mm) and by the use of an Hamilton syringe rotated on the coronal plate of about 3° from the orthogonal position, as previously described (15). Mice are euthanized 20 min after the ICV injection of luciferin (75 μg/3 μl saline). Brains are rapidly dissected and sectioned by means of a “brain matrix” (Brain Matrix, Adult Mouse, coronal and sagittal, 1 mm spacing; Ted Pella, Redding, CA) and sections are immediately visualized by the CCD camera. Gray-scale images of the sections are first taken with dimmed light. Photon emission is measured in a 15-min exposure time. Pseudocolor images associated to photon emission are generated by the appropriate image processor and transferred via video cable to a PCI frame grabber. For co-localization of the bioluminescent photon emission, gray-scale and pseudo-color images are merged. Luminescence of the brain slices is expressed as the integration of photon emission per time unit (cts/s); to be able to compare the extent of photon emission in brain nuclei characterized by a different area, data are expressed as cts/s/mm2. Photon emission in selected brain areas is quantified by means of a grid generated with the aid of a brain atlas (Fig. 20.3).
The work done in the laboratory is supported by grants from the European Community (STREP EWA LSHM-CT-2005-518245; NoE EMIL LSHC-CT-2004-503569; NoE DIMI LSHB-CT-2005-512146; IP CRESCENDO LSHM-CT-2005-018652) and by NIH (RO1AG027713).
1To validate the faithfulness of the reporter of interest, it is advisable to evaluate its activity in parallel with the activity of endogenous genes. However, differences in the spatio-temporal accumulation of the reporter and the products of the endogenous genes may be expected due to the fact that all endogenous genes are under the control of complex promoters which may influence the overall transcription rate of the reporter differentially; in addition, the turnover rate of the endogenous gene may differ from the reporter: therefore a time-course experiment must be carried out.
2A further step necessary to validate the activity of the reporter consists in immunocytochemical studies aimed at demonstrating that the molecules active on the reporter co-localize with the product of the reporter gene.