What is the optimal time for transfection efficiency measuring? - (Nov/23/2013 )
Dear Colleagues, I have a simple question: what is the optimal time for lentivirus transduction efficiency measuring (eg. by GFP)?
Transducted cells need not so much time to express the GFP protein, but why in most cases researchers measure the efficiency after 12-48-72 h., but not in 6 h., for example? Why we need to wait so long?? Mammalian cells, which was not transducted, divided approximately 4-8 times in this long time, for this reason the efficiency of transducted cells in % will be less after 72 h. then after 6 h., am I wright?
SO, what is the logic in choosing such time points?
As always - it depends. The transduction procedure and overexpression of genes can lead to stress responses. Transduction can be toxic (depending on auxiliaries like polybrene), number/efficiency of virus (MOI) and transduction method (spinoculation). Generally, cells need time to "calm down" and recover. Also the cell type and the overexpressed gene itself have some influence (length of bp, function, localisation ...). It can also happen that the overexpressed protein doesn't fold properly and triggers unfolded stress response what further affects cell growth and viability. So checking for transduction efficiency (e.g. FACS analysis) should be carried out at different time points until it doesn’t change much. My experience is that a GFP reporter develops rather quick (12h) while RFP needs more time (48h), yet as mentioned before, that all depends on other factors like cell line, introduced genes etc. I always check transduction efficiency after 24 hours and then by chance (e.g. when splitting the cells).