stable 3t3l1 transfection:serious help needed!!! - (Mar/17/2009 )
memo on Mar 25 2009, 05:13 PM said:
Hi I don't have a lot of time on my hands at the moment, but I see you have get a lot answers.
I prefer to do a single plasmid transfection instead of co-transfection as you mentioned the problem which could arise.
I transfect 3T3-NIH cells stably with a vector containing also the GFP gene. After stabilization (96-well selection) I did get a lot of resistant clones with G418 resitance, but no GFP expression. Because I could screen a lot of clones there were also positive clones in my experiment, giving 80-95% GFP (strange????) expression!
I prefer to do a single plasmid transfection instead of co-transfection as you mentioned the problem which could arise.
I transfect 3T3-NIH cells stably with a vector containing also the GFP gene. After stabilization (96-well selection) I did get a lot of resistant clones with G418 resitance, but no GFP expression. Because I could screen a lot of clones there were also positive clones in my experiment, giving 80-95% GFP (strange????) expression!
yes! i really thought that would be smarter, so this is the first thing i m planning to try . that s to clone my gene in pIRES2 EGFP. i m doing a killing curve actually and u re right about those crazy resistant cells! i mean a cell has its reasons.
i ll make sure to screen hundreds and hundreds of clones then!
so thanks for the input! and good for u that u had such high expression levels all in all!
have a great rest of the week and again thank u for ur help!
-drevilette_R-