false positive clones URGENT - resitant to G418 but not expressing protein (Mar/28/2007 )
Hey, I tried transfecting A549 and establish a stable line.
24hrs after transfection I started selecting with 500 µg/ml G418 (which I have tested before), cells started dying after appr. 4 ds, which was apparently too slow for my boss, so I increased the dose to 1000µg/ml. I got very nice clones which I expanded but when checking for expression they all turned out to be false positives.
Now my question, how could they get resistant without being transfected??? Please help cause I really need some clues soooo very urgently (actually tomorrow morning 
) Oh, resistance and insert have two seperate promotors which both are intact, so it is not a matter of the construct. But what other possibilities are there and how do they work???
Thanks
Maybe you don't actually have the insert you thought you had.
24hrs after transfection I started selecting with 500 µg/ml G418 (which I have tested before), cells started dying after appr. 4 ds, which was apparently too slow for my boss, so I increased the dose to 1000µg/ml. I got very nice clones which I expanded but when checking for expression they all turned out to be false positives.
Now my question, how could they get resistant without being transfected??? Please help cause I really need some clues soooo very urgently (actually tomorrow morning
) Oh, resistance and insert have two seperate promotors which both are intact, so it is not a matter of the construct. But what other possibilities are there and how do they work???Thanks
I am not familiar with A549 cells but 4 days to kill all non-transfectants seems to be not too slow, some select for 2 to 3 weeks; my experience in selection against G418 is that it is better to use a low conc of G418 with a long-term selection than a high conc with quick selection;
in your selection, were all non-transfectants killed, or were some left?
anyway, if you talk about expression, do you check the resistant factor or your protein of interest?
there may happen recombinations during integration into the genome which may lead to delocalization of resistant factor gene and gene of interest
I have my protein of interest in a FLAG-vector and I detect against the FLAG-tag with Immunofluorescence. However I could not get any positive result, there was only background staining. Fixation and everything was fine though. Checked that with another AB against an endogenous protein and it looked brilliant.
So after that my boss decided that all of the surviving colonies, which survived in the selection medium for quite a while (~ 5 weeks), are non transfected cell. But my questions is still: why did they survive??? 
i meet the same problem three times when i using G4181000ug/ml to select the transfected cells. I got nice clone but no interested protein was detected when expanding culture. I think the problem is the concentration of G418 is too high, so only Neo gene was intergrated into the genome under this pressure, the interested gene is then lost .
do they have anything for their tranformation that uses G418?
may you swith to an other selection antibiotic ?
Have you tried to test what is the minimum concentration of G418 to kill ALL untransfected cells (doing a killing curve, serial dilution...) 1mg/ml of G418 is not that high. Some cells can be very resistant. For A431 and a few other cell lines I had to use 1.5-2mg/ml of G418 before.
But I also agree with fred. G418 is really not very superior to use as selection marker, they are just most common nowadays. May need to look for another antibiotic.
I had this exact same problem, and I hope that you have sorted it out as I was never able to. My conclusion was that since resistance and the gene of interest were controlled by different promoters the cells only had to translate neomycin to survive so there was no need to translate the protein which is what bobxiang4 suggested, but I am curious as to whether or not you solved this problem.
Well I did, the clones were positive but were just expressing minimal amounts of the protein and therefore didn't stain well. 
for g418 selection, just follow the protocol. you should use the lower amount g418 wichi can kill all of cells within 1-2 weeks. High amount g418 and killed the cells ASAP does not mean good.
if you transfect the cells with circular plasmids, and they will be randomly cutted and then integrated into the genome. the longer of your inserts, the higher possibility the inserts to be cutted. you can linearized your plasmid outside the insert, g418 and promoter region and then do transfection. and sometimes you need select single cell clone to get cells with highest expression of your interest although it is time consuming.