the vector for stable transfection - (Feb/27/2004 )
now i am using pEGFPC3 to transfect HepG2 cells and i want to isolate stable transfectants. i can see fluorescence 24-48 hs after transfection. After i add G418 for 10 days, colonies of resistant cells begin growing. But it is so pity that the resistant clones do not give off any fluorescence!!
so i do not know if these clones are true positive clones (since there are no fluorescence)and i don't know how to do now. should i throw away these clones and do another transfection ? Maybe i can do some western blots to know if the gene has integrated into the genome and began to express ? can i isolate the genomic DNA and do some PCR to confirm this?
some people told me that pEGFPC3 is not very suitable for stable transfection and they said that pcDNA3.1 is better. Is it ture?
you can definitely do a pcr to confirm the presence of your DNA in your transfectants, you'd have to use plasmid specific primers, so that you don't get a signal from anything cross reacting in the genome. Or try one plasmid specific and one gene specific primer. You can use sequencing primers most of the time (I'm not sure if your vector is routinely used for cloning?), just check that they are compatible with each other (ie have similar Tms).
As far as I know it doesn't matter what vector you use for transfection, but one thing you can if your transfectants turn out to be false positives, is to increase you concentration of G418 when first isolating resistants, a response curve might help, concentrations required to kill different cell lines is different.
Perhaps your transfectants have incoporated the Neomycin gene into the genome but left out the EGFP. It is just a thought....
I am also venturing into this area and would be happy to help and exchange pointers.
GFP protein doesn't last long, some people say it lasts 10 days, others say one month. Please check your gene expression first before you throw away these colonies.