Advances in transgenic technology - Principles of Gene Manipulation - page 252

FK- 506 Cyclosporin A

Specific interactions

Immunophilin C Cyclophilin

I

Synthetic dimer FK-506/Cyclosporin A

Chimeric constructs

Immunophilin GAL4 Cyclophilin

Immunophilin GAL4 Cyclophilin VP16

P P

GAL4 I VP16 C

Fig. 13.4 Overview of chemically
induced dimerization between the
synthetic FK-506/cyclosporin A
conjugate and fusion proteins
containing immunophilin and
cyclosporin domains. Dimerization
assembles a functional transcription
factor that can activate a promoter with
GAL4 response elements.

- CID + CID

I I

C

GAL4 GAL4 VP16

Target gene Target gene

P P

Since this homodimer can also recruit non-
productive combinations (e.g. two GAL4 fusions),
an improved system has been developed using an
artificial heterodimer specific for two different
immunophilins (Belshaw et al. 1996). In this case,
FK-506 was linked to cyclosporin A, a drug that
binds specifically to a distinct target, cyclophilin.
This heterodimer was shown to effectively assemble
a transcription factor comprising an FKBP12- GAL4
fusion and a cyclophilin-VP16 fusion, resulting
in strong and specific activation of a reporter gene
in mammalian cells (Fig. 13.4). A more versatile
system has been developed that exploits the ability
of another immunosupressant drug, rapamycin, to
mediate the heterodimerization of FKBP12 and a
kinase known as FRAP (Rivera et al. 1996). In this
system, FKBP12 and FRAP are each expressed as
fusions with the components of a functional tran-
scription factor. In the absence of rapamycin there
is no interaction between these fusions, but when
the drug is supplied they assemble into a hybrid

transcription factor that can activate transgene
expression. Transgenic mice containing a growth-
hormone gene controlled by a CID-regulated pro-
moter showed no expression in the absence of the
inducer, but high levels of human growth hormone
24 h after induction with rapamycin (Magari et al.
1997). The advantage of this system is that rapamycin
is rapidly taken up by cells in vivo , and it decays
rapidly. The major disadvantage of immunosup-
pressant drugs as chemical inducers of dimerization
is their pharmacological side-effects. An active area
of current research is the design of modified CID
systems that do not interact with endogenous
targets in the immune system, and thus provide
rapid and effective transgene induction with no
side-effects (Liberles et al. 1997).

Inducible protein activity

The inducible expression systems discussed above
are all regulated at the level of transcription, such