transient transfection - (Oct/27/2007 )
Hi
Could someone explain me why almost 99% of transiently transfected cells with whatever GFP are not fluorescent during division while the rest of the cells, stages- is. I'll be really glad if you can suggest me what to do to have dividing cells fluorescent without preparation of stably expressed line.
cheers
sedzia
Could someone explain me why almost 99% of transiently transfected cells with whatever GFP are not fluorescent during division while the rest of the cells, stages- is. I'll be really glad if you can suggest me what to do to have dividing cells fluorescent without preparation of stably expressed line.
cheers
sedzia
I am not sure how certain it is for your claim. EGFP has a pretty long t1/2 and it should be carried over into various cell cycles once expressed. Tell us how did you come to this conculsion? I know for sure that stable transfected cells does not have this problem.
Could someone explain me why almost 99% of transiently transfected cells with whatever GFP are not fluorescent during division while the rest of the cells, stages- is. I'll be really glad if you can suggest me what to do to have dividing cells fluorescent without preparation of stably expressed line.
cheers
sedzia
I am not sure how certain it is for your claim. EGFP has a pretty long t1/2 and it should be carried over into various cell cycles once expressed. Tell us how did you come to this conculsion? I know for sure that stable transfected cells does not have this problem.
hi
Maybe I was not precised. What I'm trying to do is to look at the localization of different proteins during cytokinesis, therefore I'm transfecting cells with selected protein coupled to GFP, standard plasmid transfection. The problem is that although the transfection efficiency is high only couple of cells are fluorescent being in cytokinesis stage, e.g. anaphase. It looks like that cells are loosing the construct during division. Should I add selection marker to the medium to force cells not to loose plasmid during division?
It maybe complicated and cell type dependent.
Maybe you just havent found the ones that entering the cell cylce.
I have seen cell replication after transfection in K562 cells. This cell type can not be transfected very efficiently. So you see a few cells in a view field that is positive for EGFP to begin with after transfection. If you let cells to grow for several days, you can see 4-cell and 8-cell cluster, all EGFP positive, apparently daughter cells from the one that was transfected, in a background that most are not positive.
I've also seen a video clip on the web showing time lapped cell division process ( HeLa cells) after CaPO4 transfection. It clearly shows cells went through various stages of cell cycle. So it is possible to catch these events.
However, you muct also know that bulk of the DNA transfected/taken up by cells went degraded, whcih will generate a lot of dsDNA fragement, typical DNA damage signal that in some cell types may become a strong signial for cells to halt the cell cycle untill DNA damage is repaired. Do check if this a possibility. You may need stable transfection or viral infection to get around with this problem.
Could someone explain me why almost 99% of transiently transfected cells with whatever GFP are not fluorescent during division while the rest of the cells, stages- is. I'll be really glad if you can suggest me what to do to have dividing cells fluorescent without preparation of stably expressed line.
cheers
sedzia
I am not sure how certain it is for your claim. EGFP has a pretty long t1/2 and it should be carried over into various cell cycles once expressed. Tell us how did you come to this conculsion? I know for sure that stable transfected cells does not have this problem.
hi
Maybe I was not precised. What I'm trying to do is to look at the localization of different proteins during cytokinesis, therefore I'm transfecting cells with selected protein coupled to GFP, standard plasmid transfection. The problem is that although the transfection efficiency is high only couple of cells are fluorescent being in cytokinesis stage, e.g. anaphase. It looks like that cells are loosing the construct during division. Should I add selection marker to the medium to force cells not to loose plasmid during division?
do you expect expression of your EGFP-tagged protein during mitosis? try to varify with stable transfected cells where the EGFP-tagged protein expression should start again after cell division