I have created stable cell line using MEF cells and selected them using Zeosin. However Immunoflouresent experiment using confocal revealed that only about 5% cells are expressing the gene of interest. What is the best method to select the clones with my interested gene?
This is not really my field of study, but I believe FACS (Fluorescence-activated cell sorting) is a good method to isolate cells that you can label with a fluorophore.
This is a common problem when your gene of interest and your antibiotic selection cassette are expressed from different promoters. Resistant cells that are negative for your transgene have either silenced the promoter for that gene or simply express extremely low levels that will be below the detection limit for directly coupled fluorophores like GFP, or occasionally by indirect IF.
As Ilya suggests, one option is to perform FACS, if possible, for your gene of interest. Another is to redesign your transgene expression cassette such that it is under the same promoter as the antibiotic resistance gene (ie. design GOI-IRES-ZeoR, or GOI-T2A-ZeoR cassettes and repeat the stable cell line selection).
I agree with Daniel, use an IRES seq to have a single mRNA for you GOI and resistance. The extra work is worth it!
Good luck
- Badges
- Science topic