After successful transduction of airway epithelial cells with the gene of interest we apply puromycin continuously in the culture medium in our lab. It is possible that using this continuus selection pressure the cells decrease the expression of the gene of interest while stay resistant to puromycin?
Im not sure what construct you are using but I have experience with the pINDUCER system which has a double cassette; one for doxycyline induction and one for selection. I find that increasing the selection pressure, keeps the cell population atleast 90% positive (cells that have been successfully infected). This has no effect on the transduction of the gene because it is driven by a different promoter in the construct. Im not sure if this answers your question, because I dont know what system or construct you are using.
Yes it's possible if the ectopic protein that you express is degraded. You can check that by treating you're cells with MG132 (proteasome inhibitor) and look at the protein level if the expression is increased.
Yes they do. Actually, if you want to use those cells in experiments you should leave them first a couple of days up to a week without the antibiotic.
It is possible that one gene gets silenced or recombined. If you want to be ABSOLUTELY sure you have expression with resistance you may use a fusion protein (your_protein-2A_peptide-puro) that will self-cleave once expressed.
It is sort of like IRES but on the level of protein ;)
See here:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018556
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