Transfection but no expression - (Oct/21/2013 )
I would really appreciate your opinion about this problem I've faced.
I cloned into PcDNA 3.1 Hygromycin and Zeomycin. The insert sequence as well as the promoter regions were sequenced and they're perfect!
I transfect these vector into Mouse embrionic Fibroblast using Lipofectamine LTX that is quite good for transfect sensitive cells. The transfection rate was stablished at 1:7
Five days after transfection we start the selection.
I ran PCR and the cells were positive as well as they're growing pretty fine under the presence of the highest antibiotic concentration.
On this way that should be expected that these cells would express high levels of my protein but when we screen the cells by FACS they are all negative.
I haven't tested in Western Blotting but anyway they should be positive in FACS.
I would really like your comments for this problem
Can you still run a WB, just to be on the safe side ? Because i once had the trouble that WB and FACS results were not consistent with each other...
For FACS the antibody needs to be one that recognizes the native epitope - many antibodies will only recognize the denatured epitope - check your antibody datasheet.
Do you have a + control for the FACS, - i.e. an antibody that you know works?
thanks for your replying.
Yes I have positive controls for FACS that are cells transfected in the past with the same gene. I don´t know what´s going on now that's not working.
I use this antibody: http://www.abdserotec.com/mouse-cd51-antibody-rmv-7-mca2461a488t.html
I may have forgotten to mention that these cells are deficient of these genes and we´re trying to rescue them.
Two things of note - it is unusual to start selection 5 days after the transfection - by this time many of the cells will have lost the plasmid - growth under selective pressure doesn't mean that the plasmid is inserted! It could mean that you are using too low a selection level (run a titration before you start transfecting), or it could mean that your antibiotic has gone off, or it could be that the cells are too confluent for the selection to work properly (for most drugs like hygromycin, and zeocin the cells must must be kept below 70% confluent for selection to work).
Checkin for the presence of the insert by PCR may be problematic if the plasmid is not integrated but still present, but Southern blotting is best - works well for genomic insertion.