inducible mammalian expression vector

dirk dobbelaere dobbelaere at ipa.unibe.ch
Fri Nov 11 05:54:11 EST 1994


Hello transfecting Netters!

We are working on T-cells that are transformed by infection with an
intracellular parasite.  Infected cells acquire the ability to proliferate
continuously.  We are examining the different T-cell signal transduction
pathways which could be the target of parasite interference.  In order to
establish if a particular pathway plays an essential role in the
transformation process, we would like to make use of either antisense RNA
expression or the overexpression of dominant negative mutants that could
block components of the pathways that we think are involved.  In  order to
rescue the transfectants, propagate and clone them, it is essential that
the inhibitory antisense RNA or dominant negative mutant is only expressed
upon induction.   

Our experience with antisense RNA expression by constructs that are under
the control of a cadmium-inducible metallothionein IIA promotor was
satisfactory in transient experiments, but lousy in permanent
transfectants.  In our hands, the system was very "leaky" and induction
bad, resulting in considerable inhibition of growth without induction, only
limited additional inhibition upon induction and very bad cloning
efficiency of the antisense transfectants.  

Our cells also do not respond to dexamethasone, making an MMTV promotor,
which was our first choice, useless.  

In other words, we are frantically looking for other reliable inducible
systems in which expression of dominant negative mutants or antisense RNA
can be tightly controlled, allowing us to monitor the inhibitory effects in
detail.

I would be grateful for good suggestions and even more grateful if someone
could offer me a suitable construct (would be considered a collaboration
with co-authorship guaranteed).

cheers!



More information about the Methods mailing list