Hi all,
I'm working with a cell line that was transfected with a plasmid coding for hygromycin resistance in an attempt to generate a stable cell line. I noticed that when I passaged cells using medium supplemented with non-lethal concentration of hygromycin, the cells would perish. However, if I allowed cells to recover in non-supplemented medium and then treated them with the same concentration of hygromycin, the cells would not die. Can anyone suggest reasons why the cells would be sensitive to the antibiotic during passaging?
Thanks!
This is just my assumption.
During passaging, when treated with trypsin etc the cell membrane is fragile, and the penetration of the hygromycin is higher. However, when you let the cell recover first, then the cell membrane etc are intact and cells have completed the cell cycle and ready to resist the treatment.
To confirm this, check the amount of cell death after cells have recovered and treated, if its less than say 20% then my theory supports it, as usually less than 20% of the cells are undergoing mitosis at any given time.
Hope this helps you to get thinking, also please update what you find, will be interesting to know for sure.
i partially agree with the answer above but the following needs to be considered: Hygromycin B inhibits protein synthesis. Passaging indeed is stressful and this likely impacts (transiently) the expression of HygB resistance gene (the kinase) and its level drops to below the level, sustainable for maintaining the selection. This seems identical to situation when you transfect, then have to wait 2-3 days (for protein to be produced from mRNA, expressed from your transgene), and only then apply the selection drug. Maybe you passaging in a way, which causes cell damage. However, normally if you have a stable transfected clones, you don't need to remove the selection drug during passaging. Based on your comment "working with a cell line that was transfected ... in an attempt to generate a stable cell line" you have not attempted to generate stable colonies and passaging too quickly after transfection. Some colonies just die (it's normal) because a number of loci (where your transgene is integrated) are gradually silenced by nearby chromatin. This takes time, from 2-3 days to a week or longer. So you have a situation where you have emerging colonies resistant to HygB, but then the expression of the drug-resistant gene goes down progressively, and as you passage, culture dies. I suggest to establish colonies with robust long-term growth (2-3 weeks) before passaging to select for colonies, where Tg expression is stable indeed. The drug level can be reduced for routine culture after such colonies are established. Mixing many colonies by bulk-passaging the culture is not a good idea, as you will have variability in Tg expression in different cells (progeny of the original colonies)
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