I am looking for a cell line which doesn't show constitutive activation of the PI3K-Akt pathway. I am planning to do a protein interaction study using activators of this pathway like Insulin, hence I need a cell line which won't show constitutive Akt activity. Another criterion is transfection efficiency. Can someone suggest a good cell line for my intended study?
I have been using HuH7 cells, and from what I have seen they don't show constitutive Akt activity. They respond to very low concentrations of insulin (1nM), and I have had good results transfecting them with Lipofectamine.
Dear Aditya
The main problem that I imagine you face is that you see high levels of phospho AKT as a read out by western blotting. This usually comes from two sources:
Either your cell line has a mutation or loss of PTEN, which means that you have high basal levels of signaling through PI3K/AKT. You can just check this with a simple PTEN western to see if you have expression (However expression will only tell you that you have not got a deletion of PTEN). The second answer is probably that you have high levels of signalling through receptor tyrosine kinases due to the growth factors in your media, and the best way to reduce this is to titrate the serum concentration that you use in your experiments to a point where you have low base line activation of the pathway without adversely effecting the cells health or viability. That way when you come to add growth factor you should see an increase in PI3K/AKT activation which you can directly attribute to insulin, and even do dose response experiments. IGF-1 is a really good activator of PI3K/AKT and GroPep make a truncated form for cell culture which is not blocked by IGF binding proteins.
I often use UMR-106 cells which are a rat osteoblast like osteosarcoma cell line which have normal PTEN and respond really well to IGF-1 when serum deprived. These cells grow fast and transfect well with Effectene from Qiagen, but other reagents work as well.
Thanks a lot Rose and Andrew.... I would prefer Huh7 cells, as they are more relevant to our study..
skeletal mucle cell line (L6). they do respond to insulin and the akt pathway is inducible
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