The new technique, described in the open access journal BMC Biotechnology, will allow detailed analyses of correlations between behavior, gene expression and aging.
John Tower led a team of researchers from the University of Southern California, Los Angeles, who carried out the fluorescent experiments in Drosophila flies. When the flies are illuminated with blue light, the authors' video tracking system allows tissue-specific GFP expression to be visualized, then quantified and correlated with 3D animal movement in real time. According to Tower, "These methods allow specific temporal patterns of gene expression to be correlated with temporal patterns of animal activity, behavior and mortality".
The green fluorescent protein gene is isolated from the jellyfish Aequorea victoria and encodes a protein that absorbs blue light and emits green light. When a fly expressing GFP is illuminated by blue LEDs, filtered cameras can detect the green fluorescence that results and the fly's movement can be tracked at a rate of 60 frames per second. By linking the expression of GFP to the expression of other reporter genes, it is possible to determine when these genes are on or off, and how this is associated with a fly's behavior.
Tower said, "A large number of strains exist where GFP or some other auto-fluorescent protein is used as a reporter for specific gene expression in Drosophila and other organisms. Our methods should be readily adaptable to such reagents, for example we have recently been successful in tracking DsRED fluorescent flies".
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The investigators then tested their pieces of esophageal lining in whole animals. When the tissue-engineered patches were transplanted under the skin of immunodeficient mice, the cells formed epithelial structures. Additionally, in a mouse model of injury of the esophagus in a normal mouse, which mimics what happens during acid reflux, green-stained stem cells migrated to the injured lining cells and co-labeled with the repaired cells, indicating involvement of the stem cells in tissue repair and regeneration.
Eventually the researchers will develop genetically engineered mouse models to be able to track molecular markers of esophageal stem cells found in a micorarray study. The group has already developed a library of human esophageal cell lines and is looking for human versions of markers already identified in mice.
"The ultimate goal is to identify esophageal stem cells in a patient, grow the patient's own stem cells, and inject them locally to replace diseased tissue with normal lining," says Rustgi.
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