8 replies to this topic

[ulka]

I am  trying to do a stable transfection in mammary breast cell lines and have had problems in getting successful transfections. I am using lipid based transfection protocol. I cotransfect the cell line with the vector of the gene of my interest. It has a CMV promoter . I use puromycin as a selsction marker and I co-transfect my cells with the plasmid that has the puromycin gene. I'd like to seek help, as the protocol I'm currently using does not seem to be working well.

[IsabelleM]

What kind of problem ? Is it that all cells die, or you don't seem to get any colony ? I never transfected in mammary gland cell line (such as ZR-75-1, or MCF-7), but i did a lot in HK293. I used pcDNA vectors, with G-418 or zeocyn resistance. Normally, i do a pre-test on the effect of the antibiotic on the cells. I use different concentrations to see at which point and how much time it takes to my antibiotic to have a complete effect on the cells. I can tell you that zeocyn has a stronger effect than G-418, and is much faster. Maybe you are using a too strong concentration of antibiotic. I transfect in 6 well plates, and i normally see distinct "colonies" of stable transfected cells after two weeks. Each colony is selected with a sterile stainless ring, with sterile siicone, in which i put a dose of trypsin. Every colony is passed in a well (24 well plate). Sometimes, i pool 3-6 colonies, but you usually get less activity in these pools than distinct colony.

[ulka]

Thanks. My problem is that after the selection is done, I do not get activity in the selected cells. I have a fluorescence tag and  check activity using  a fluoresence microscope.
So I think the problem lies in both transfection itself where i do not get plasmid integration and also in selection.
I allow 48 hours of selection after transfection as this is the time taken for the control cells to die after I add puromycin.This is what I got when I did the death response experiment.
I use a CMV promoter driven plasmid. I wonder if CMV promoter is good for creating stable cell lines for the ones I am using.

[IsabelleM]

Are you using a marker for the efficiency of the transfection itself ?

[ulka]

I am currently not using a positive control but I know of people who have done transfections in this cell line.

[IsabelleM]

Maybe you can find a marker in your lab that you can co-transfect, because it is possible that the transfection is the problem. I did a lot of stable and transcient transfections, and i do a positive control everytime. I can tell you, that even if i use the same quantities, or the same protocol, there are differences, sometimes important, in the efficiency of the transfection. It may be due to lipofectine (or any system) or the cells

[ulka]

Thank you. I'll try doing that this time.

[IsabelleM]

Like you said, it would be important to verify if vectors such as pcDNA or others recombinants wouldn't be more efficient. I know that "old" vectors such as PBK-CMV are very poor vectors for transfection. Another thing, how do you purify your DNA ? We use cesium chloride purification, and we saw that it is more efficient than commercial kits or DNA purification (i know, it takes a lot of time, but it is worth it)

[ulka]

We also use CsCl purification protocol. So I guess that should not be a problem.
I am also trying to find if CMV vectors are good systems for epithelial cells but have not been able to find anything conclusive, though some references do seem to be using CMV driven systems.