I have generated lentiviral mediated gene overexpression in fibroblast cells. I treated cells with puromycin (10 ug/ml) for 28 days and observe GFP (expressed as a reporter gene along with therapeutic gene) positive cells under the fluorescent microscope. All cells were GFP positive at the end of treatment. When I stop puromycin treatment, GFP intensity and the therapeutic gene expression levels (confirmed by RT-PCR) started to decrease. Should I go on puromycin treatment during the main analysis such as cell viability, cell cycle analysis or cell migration? How can I achieve stable therapeutic gene expressing fibroblast to conduct all experiments?
Thank you,
Although the lentiviral vector should integrate you still need to provide selective pressure to maintain consistent expression. You can decrease the concentration of puromycin after selection to say ~2ug/ml, but you should maintain the cells in the antibiotic.
It is important that you continue to use puromycin for selection purposes. This helps to rule out any doubt about potential escape clones when analyzing data using these cells. As Richard suggests, I would reduce the concentration of puromycin used during maintenance.
When we worked with cells silenced by lentiviral vectors, we did not perform experiments in the presence of puromycin, but rather performed two selections before proceeding.
Thank you for your answers. I have been told that I should use clonal selection? So, is it necessary for gene function analysis experiment?
Cloning can help you to select a clone with best expression of GFP and maybe with reduced silencing. Silencing can be a problem also if the gene is stable integrated in the genome. How quickly is decreasing the GFP signal? Do you will theoretically have time to perform your experiment without puromycine and without loosing gene expression? If not, you probably have to maintain the cell in puromycine in reduced concentration also during experiment to ensure expression of GOI...
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