EGRE3

1
Ecdysteroid-regulated transgene induction
Ecdysteroid
VP16
(A)
Co-repressor
RXRa
(B)
VgEcR
EGRE3
Binding of the ecdysone receptor (VgEcR) to its
ligand (A) results in the formation of a
heterodimeric complex with RXRa thereby
initiating an induction of transcription (B).
2
A
B
C
Construction of the mammary targeted inducible
mice. The Enhancer line (A) was mated to the
Mammary Receptor line (B) to produce the
Inducible Reporter line (C) Addiion of ligand
will induce B-Gal Activity in the mammary gland,
exclusively. (Albanese, et al., FASEB, 2000)
3
Addition of the ecdysteroid, ponasterone A,
resulted in induction in b-Gal activity
exclusively in the mammary gland (with only
low-level activity activity in the salivary
gland) within 24 hours of administration
(Albanese, et al., 2000).
4
Advantages of Ecdysteroids

• High purity

• Rapid induction

• Sustained expression

Albanese, et al., FASEB 2000
5
Light-inducible Transgene Induction
A
B
Caged (inactive) Ecdysteroid
Active Ecdysteroid
VP16
Co-repressor
VgEcR
EGRE3
Caging of ecdysteroids with photo-labile
compounds renders them inactive (A).
Light-induced uncaging results in receptor
activation and initiation of transcription (B)
(Lin, Albanese, Pestell and Lawrence. Chem and
Biol. 2002, 91347-1353.)
6
Light activated gene expression
Light activated gene expression
Caged ecdysone(butylstannylene alkylation with
bromo-methyl nitro-benzene)
A) Caging of ecdysone. B). Time dependent
induction of transcription following cuncaging
(illumination) (Lin, Albanese, Pestell and
Lawrence, 2002)
illuminated
Washed and illuminated
Control
7
Light activated gene expression
Unmodified Ecdysone
Uncaged Ecdysone
Caged Ecdysone
Immunohistochemistry showing induction of
transcription of A) Unmodified ecdysone, B)
Uncaged ecdysone and C) Caged ecdyson. (Lin,
Albanese, Pestell and Lawrence, 2002)