Generation Of Reporter Mice Research Articles

Pericyte Mapping in Cerebral Slices with the Far-red Fluorophore TO-PRO-3.

This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.

No Zones Left Behind: Democratic Hepatocytes Contribute to Liver Homeostasis and Repair.

Despite minimal turnover, liver cells possess immense regenerative capacity. Some studies suggest existence of a hepatocyte subset with such unique capabilities. However, in the current issue of Cell Stem Cell, three independent studies (Chen etal., 2020; Matsumoto etal., 2020, and Sun etal., 2020) demonstrate an equitable homeostatic and reparative potential of all hepatocytes, irrespective of their lobular location or ploidy status.

Reporter mice for the study of long-term effects of drugs and toxic compounds.

Noninvasive, imaging-based methodologies provide for the first time the possibility to spatio-temporally investigate physiopathological events and long-term effects of drug administration of exposure to environmental and alimentary toxic compounds. Hence, this novel methodology could enable us to measure the dynamics of specific molecular pathways in live animals. In the last few years, animals, particularly mice, were genetically modified to respond to a specific stimulus with the production of proteins, named "reporters," that are easily detected and quantitated by in vivo and ex vivo imaging. These "reporter mice" are gradually being applied to the pharmaco-toxicological research. In the generation of a reporter mouse useful for pharmaco-toxicological studies several elements need to be considered in the selection of the reporter system: the half-life of proteins should be compatible with the necessities of the study to assess the onset and the termination of the stimulus of interest, in all tissues the response should be proportional to the given stimulus, and the imaging modalities requested for reporter measurements should be applicable to high-throughput screening. Bioluminescence-based imaging (BLI) in small animals has the advantage over other modalities that does not require too sophisticated equipment or specifically and highly trained personnel, and furthermore may be carried out at a relative rapidity and low cost; for these reasons several luciferases have been developed for in vivo imaging applications and used in the generation of reporter mice. We here describe a BLI-based reporter mouse created to respond to estrogenic stimuli, which has been applied to the study of female physiopathology as well as for the identification of the effects of selective drugs or toxic compounds present in the environment and in the alimentary chain.

Cancer modeling: Modern imaging applications in the generation of novel animal model systems to study cancer progression and therapy

Cancer is the result of a series of genetic and epigenetic mutations that evolve over years even decades and lead to the transformed phenotype. Paradoxically, most methods developed to study these changes are static and do not provide insights on the dynamics of the sequela of steps involved in tumorigenesis. This major shortcoming now can be overcome with the application of reporter genes and imaging technologies, which are providing tools to examine specific molecular events and their role in the carcinogenic process in single cells. In the last decade reporter-based biosensors were created to study gene transcription, protein/protein interactions, sub-cellular trafficking and protease activities; this wealth of systems enable to monitor intracellular signaling pathways at several key check points specifically involved in cancer cell development. The challenge is now to extend cell-based models to the generation of reporter mice, where non-invasive in vivo imaging technologies allow to follow single molecular events. When combined with murine models of cancer, these technologies will give an unprecedented opportunity to spatio-temporally investigate the molecular events resulting in neoplasia. The aim of the present review is to highlight the major changes occurring in this rapidly evolving field and their potential for increasing our knowledge in cancer biology and for the research of novel and more efficacious therapies.