Is it a general effect that stably transfected cell line has lower cell prolifer - (Jul/17/2008 )
Hi, everyone. I have established a stably transfected cell line with my gene of interest. I also have a control cell line which is the empty vector containing a Flag tag and carrying a Neo resistant gene. I have just recently found that the stably transfected cell line with gene of interest has lower total protein amount, around 20% decrease compared to the control, and initially I seeded the same amount of both cells and harvested them at the same time. I am thus wondering if this is normal since stably transfected cells have some stress because of the integrated exogenous gene, so they grow slower? But my control cell line is also integrated with Flag protein though small. So is it a gene-specific effect? I treated both cells with G418 during routine culture. Look forward to you guys' helpful suggestion! Thanks.
-Jason
Hi Jason,
No it is not a general effect. How many days did you grow the cells, and what was the density when you harvested? I ask because if the cells are very confluent, then it could be a difference in eg. contact inhibition rather than proliferation.
Anyhow, worth chasing up by counting cell numbers over a few days, as a cheap way to start. Also, have you checked that your stable cell line has high % expression? Lots of cells stop expressing the gene but remain resistant.
Thanks for your suggestion. I grew cells for 5-6 days till they are confluent then I harvested cells for assay. I am not pretty sure if it is caused by contact inhibition since when I harvested cells, I checked cells under the microscope, some spaces between cells were clearly observed in stable transfected cells but not the control. Btw, how to accurately check the % expression?
No it is not a general effect. How many days did you grow the cells, and what was the density when you harvested? I ask because if the cells are very confluent, then it could be a difference in eg. contact inhibition rather than proliferation.
Anyhow, worth chasing up by counting cell numbers over a few days, as a cheap way to start. Also, have you checked that your stable cell line has high % expression? Lots of cells stop expressing the gene but remain resistant.
To check % of cells expressing, I'd plate the cells on coverslips, do immunofluorescence with flag antibody then see the % of cells that are above background (compare with control coverslip having only secondary antibody).
Sounds like your stables may grow slower if you see spaces between them but not the controls.
-Jason
there is no general rule; it depends on the gene, where the gene was integrated (if you do not use a targeting vector), and how many copies are integrated in the genome; even various stable clones may show differences in viability and proliferation