HEK 293 Stables - (Sep/25/2006 )
Hi,
I have been working with HEK293 cells for a while and recently have been trying to generate stable cells expressing my protein of interest, unfortunately without success (using G418 selection). I have obtained transient expression of my protein of interest and have also been able to generate cells that stably express a fluorescent protein alone.
The problem I find is that HEK cells appear resistant to G418. I conducted a kill curve experiment and found that even up to 2mg/ml exposure over 3weeks (cells initially seeded at a very low density) they failed to die.
In addition my transfected cells began looking more round after G418 exposure (500ug/ml) and completely disappeared after a week (from an initial transfection efficiency of 60-80%).
Just wondering if anyone else has had similar experiences with HEK cells and if you had any words of wisdom?!
Many thanks
welcome to the club " I hate to stably transfect 293"
I also went up to 2 mg of neo and they were happily growing, youll find my experience in one tf the threads fo this forum.
I shift to HeLa and selected cells on the antibiotic but expression in these ones was completely lost, so now am trying in saos 2 cells
the reply not very encouraging I suppose 
I have been working with HEK293 cells for a while and recently have been trying to generate stable cells expressing my protein of interest, unfortunately without success (using G418 selection). I have obtained transient expression of my protein of interest and have also been able to generate cells that stably express a fluorescent protein alone.
The problem I find is that HEK cells appear resistant to G418. I conducted a kill curve experiment and found that even up to 2mg/ml exposure over 3weeks (cells initially seeded at a very low density) they failed to die.
In addition my transfected cells began looking more round after G418 exposure (500ug/ml) and completely disappeared after a week (from an initial transfection efficiency of 60-80%).
Just wondering if anyone else has had similar experiences with HEK cells and if you had any words of wisdom?!
Many thanks
I know some kind of 293 cells are G418 or hygro resistant. You may turn to other cell lines or resistant markers.
we recently got some G418 (tissue culture center), which doesnt seem to kill cells at all. We figured the G418 is inactive or some thing is way off with the conc. during preparation.
We r preparing new antibiotics ourselves and abt to do a conc. curve.
there r also 293FTs which have the G418 resistance gene so this will not work.
Native 293cells are not resistant to G418
But 293T are resistant to G418 or hygromycin i suppose, the gene used to select cells extpressing large T epitope
Seems fine to me.
We use 1.5mg/ml G418 for selection.
I observed that lots of non-transfected cells survived during the first week or so, but they die gradually whenever you repalted the cells at low density. Make sure include G418 when you plate the cells.
But 293T are resistant to G418 or hygromycin i suppose, the gene used to select cells extpressing large T epitope
Just wondering if anyone else has had similar experiences with HEK cells and if you had any words of wisdom?!
Many thanks
HEK293 cells transfected with Lipofectamine 2000. 95% efficiency. Went up to 2mg/ml G418 for selection as the PI couldn't believe the results because there was very little cell death after transfection.
Sequencing and patch-clamping confirmed the presence of our insert. Sounds like yours are just fine.
Me, I love working with them. IME, plain vanilla HEK293s transfect well, are tolerant of all sorts of abuse and work just fine apart from not being as adherent as I'd like.
Don't forget to keep a maintenance dose of G418 after selection. We used 40ug/ml. And watch for the tendency of HEK293s to lift under washing. If they do, trypsinize, spin and remove PBS and pass as per normal and they'll be fine.
b3ka