can cells just stop having the gene? - (Mar/26/2007 )
i have a 293 cells stably expressing 2 genes and i am using G418and zeocin 200ug each , i know for sure that my cells produce one protein of the 2 genes its carring but i dont know about the other one? could it just stop producing it?
how come despite using the proper antibiotic for expression and maintinance?
once stably incorporated protein expression should last. But in rare instances, the protein might not be expressed or express at very low levels.
This can be because you have 2 different cassettes, one for transgene and other for antibiotics. So something could interefere with expression of one of them and not the other.
i havent done experiments with stable line for a very long time to know if the could lose it over a period of time. I have used them for some experiments during which they behave fine and then we throw them, as we have many cell line stocks.
Some of my colleagues have tried to keep transfected cell lines for a long time. They told me that after a while, cells only keep the antibiotic resistance gene and somehow kick the gene of interest out, so that they would have no expression of their protein of interest.
The lenght of the period over witch you keep the cells may be important too.
thank you so much
i wanted to make sure about it so i seeded some cells and lysed them then did WB for my suspected protein but it turned out that my cells got it , so i have to figure out what was wrong in my exp. may be the transient transfection of my 3rd gene?