Hello everyone.
Question when you are developing stable cell line in CHOK1 parental cell.
I want to have a pure cell population expressing a specific receptor quantity at the cell surface (nice well defined single peak in cytometry analysis). After transfection and selection with G418, I sorted some clones (picked from petri dish), but the population wasn t pure after some week of growing (still some neg cells polluting the positive cells). I did another sorting and straight after a limited dilution with my positive fraction. After few days of cell growing I analysed my cell population, but I still found a mixed population (not a well defined single peak). How this can be possible? Have you ever seen this phenomena? Do you have some ideas to explain that? Do you have tricks for that? My G418 concentration is @ 600ug/ml (which is not that aberrant)
Many thanks in advance,
Alice
Hey,
it might be that the cells are silencing the receptor. It might be an idea to analyse the same population at several timepoints and check if the signal decreases in intensity.
The other thing might be your detection. Are you staining for the receptor or do you have a fluorescent tag on it? If it's an antibody staining, maybe that is not even.
Dear Alice!
You look also this article:
Article Determination of the Selection Capacity of Antibiotics for G...
3.2 Selectivity Factor of G418 and HmB on BHK-21, CHO-K1, 3T3, and HeLa Cells
Figure 2
Figure 3
3.3 Selectivity Range of G418 and HmB on BHK-21, CHO-K1, 3T3, and HeLa Cells
Note the IC50 dose а G418 and SF
It sounds like you are creating cell lines by non-targeted integration i.e. Transfection of a plasmid followed by antibiotic selection. In such cases, the gene of interest and G418 resistance marker cassettes are chopped and randomly integrated in the genome, meaning not all cells that are G418 resistant will express Gene of interest. In my experience this method is not efficient as the cells eventually lose G418. Puromycin selection works a lot better. I would suggest using Piggybac system or retroviral based method for more stable and uniform population.
Have you tried analyze your cells before and after the G418 selection? perhaps the selection should be done more aggressively (more time). You can also follow on Sumanth advice and switch to Puromycin which is well established and might generate better selection. Silencing is certainly an option, I would attempt to optimize the selection before confronting with silencing, which will require quicker handling and experiments.
Good luck
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