Heterogeneity of protein expression level in transciently transfected cells.

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Sun Aug 8 10:40:31 EST 1999


Hi Andrew,


Andrew Leung wrote:

> Hi Frank,
>     My problem is further complicated by the fact that the gene of my
> interest is guessed to be deleterious to the cells. So I think a stable cell
> line may not be established possibly. Do u think an inducible construct with
> stable transfection technique could help?
>
> Andrew

Thanks for the additional information. Indeed, with a harmful gene you will
have difficulties in makings stable clones if you use one of the simple vectors
with a constitutive promoter (like CMV). I´d suggest you clone constructs in a
tightly regulated vector and make stable cell lines then. There are some
vectors on the market  that allow for an inducible expression of your gene of
interest with no detectable basal expression, e.g. an ecdysone-inducible one
(Invitrogen) or a Tet-On system (Clontech)(no affiliation with any mentioned
company). I haven´t tried those system yet because the proteins I expressed to
date had not been harmful. So, I cannot tell you my personal experience.

Frank





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