hey guys I have a luciferin reporter genes transfected hela cells, when I first anabiotic cell and stimulate, the data comes out good, stimulation groups are 6 times of control group, but after I passage one or two times it down to 2-3 times, why does that happen? I also use selection marker.
passage:when cell grows to 70%-80%, passage them into 4 dishes.
Yes, very interesting because also incomplete : what kind of cell line you are using, what is the promoter type, that are you studing/looking at ?
I think your cells mixed up luciferase gene transfected and non-transfected Hela cells. In my lab, we do limit dilution. That is to seed one cell in one well of 96 well plate. After 2 weeks, we made one replica plate as 50% cells trasferred and left 50% cells used to luciferase assay. Using this method, you made transfected cells with various luciferase expression level. You can choose best one. This process is too tedious. However, your cells will have very stable level of luciferase expression.
First of all, the 6X expression is not too good.
Patrick had a good question, which type of promoter you use.
Which antibiotics was used as selection marker? How much antibiotics was applied to the cells? How long did you make the selection? Usually one month or longer is necessary for isolating a stable transfected line. You might have used transiently transfected cells.
Just look around in the WEB how to isolate, and propagate a stable transfected cell line.
Hi Lyla,,
Provide little more "Specific details".
The problems you face is more common,,You should screen for stable expression first prior to your experiments,,Also try to do limit dilution as Lee said. Good Luck..
I think when you determine luciferase activity the first time there are still many plasmids present in the cells. This is still transient transfection. Afterwards, with every mitosis the amount of free plasmid will go down and after some weeks of selection you end up with only a few stable integrations of the transgene per cell. Some of these inegrations will also happen in silenced parts of the genome. So it is generally expected that stable cell lines have lower expression than transiently transfected cells. Do you really need a stable cell line? Then do as suggested, and clone the cells (for example by dilution into a 96 well plate) and screen for the best expressing clone..
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