stable 293 transfectants - (Jun/20/2006 )

little problem here with stable transfection.
My cells are growing happily at 1.6 mg/ml of G418.. and they grow fast.. they are 293 cells. anyone had experience with this cells? and stable transfectants using neomycin?.
I read the other threads and found them useful.

1.6mg/ml???
never gone that up because mine 293 are dying well at 600µg/ml (ut also at 400µg/ml).
So pb maybe come from your antibiotic solution.

I can use a max. of upto 800ug/ml for my 293FTs , more kills them.

hey.. I quit selecting 293 and now I selected transfected HeLa cells at 800ug/ml.
nevertheless, once selected i didnt see any overexpression of my protein.. contruct works beatifully for transient tranfections... this was very dissapointing
800 ug/ml killed all my untransfected hela celles and my transfected are growing nice ..
I almost thought I got an overexpressing cell line that would help me not transfecting cells each time I need protein

What's your construct? if both your gene and neomycin-resistance gene has very potent promoter, such as CMV or SV40 promoter, as well as the two gene is in the same construct, you should get stable clones with G418 selection.
I transfected 293a (based on 293) and get stable ones by screening with 2mg/ml G418.

Hi little cell
Am using pcDNA with CMV promoter for my gene and sv40 ori next to my neomycin resistance gene
I selected the cells in geneticin but after screening for my protein a realized that non of my clones were overexpressing it... do you think transcription of these can be shut down by the cell? even if the resistance cassette is working fine?
thanks

Thanks! I might have to think of recloning

does your plasmid have a kozak sequence before the gene? If not, you may find it difficult to express the gene at high levels.

bob

Any chance you are working on 293T or other derivatives by accident? These are stable 293 cells with T7 polymerase and are neo-resistant.