Good evening - I need to come up with a project idea for co-op. The project needs to be mostly FACS-related but also give exposure to other techniques, such as cell culture and molecular biology techniques. I had an idea of analyzing transfected cells (established cell line and with a fluorescent reporter gene) and sorting them based on their different levels of fluorescence intensity (low, med, high). grow them up and characterize them .
- For a random integration I'm guessing the differences in fluorescence intensity variation is due to how many times the reporter gene gets integrated? (so high intensity = high copy number, and maybe therefore a greater chance for instability??
- How about for a targeted integration, what does the different levels of florescence intensity can tell us?
Hi Dana,
It sounds like an interesting project.
For both random and targeted integration, there are many possible reasons to account for variable fluorescence intensity on a cell-by-cell basis in addition to what you've already listed. For instance whether the fluorescence reporter is shut off by the cell via DNA methylation.
In either case for your project, at minimum you'd probably want to use a low MOI to infect your cells with a GFP reporter upstream of a drug resistance marker (e.g. GFP-P2A-Puro) and select for that marker.
Hi Dana,
I agree with Barry and I would to add to that the choice of cell line and reported gene can make a big difference in the success of your project. Some cells are easier to culture and transfect than others so if you are not limited to a specific cell line, maybe you can think about your choice of the cell line and the reporter gene.
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