This article describes a method for monitoring gene expression during neuronal differentiation using cortical brain slices from transgenic mouse embryos. The technique employs fluorescence and bioluminescence imaging to visualize the activity of a gene of interest fused to a luciferase reporter gene.
Start with a dish containing cortical brain slices from transgenic mouse embryos in media supplemented with luciferin.
The cortical region contains neural stem cells that express green fluorescent proteins or GFPs and a gene of interest fused to a luciferase reporter gene.
Place the dish onto the microscope stage for fluorescence and luminance measurements.
Capture test images of GFP fluorescence, focusing on regions of interest.
During neuronal differentiation, stem cells begin to express the gene of interest and produce fused proteins with the luciferase enzyme.
The luciferin interacts with luciferase enzymes, generating bioluminescence that directly reflects the expression of the gene of interest during differentiation.
Capture a fluorescence image to identify specific regions of activity, a bioluminescence image to monitor dynamic gene expression and a bright-field image to provide morphological information.
Acquire these three-dimensional time-lapse images to visualize gene expression during neuronal differentiation within the native tissue environment.
To visualize the luciferase reporter expression within the slice cultures, select the 40x objective. And place the sample dish onto the microscope stage. Acquire a test image of the fluorescence. And set the position and focus plane to the region of interest under the illumination of the appropriate excitation light. Then run the time lapse acquisition by three-dimensional luminescence, fluorescence, and brightfield acquisitions for 24 hours.
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