of the Max - MDC

Structure of the Group
Group Leader
Dr. Manfred Gossen
Scientists
Dr. Mathias Hampf*
Graduate and Undergraduate
Students
Vishal Agrawal
Daniel Bauer*
Ibrahim Kocman*
Tina Baldinger*
Technical Assistants
Marion Papst
Secretariat
Petra Haink
* part of the period reported
Drosophila ORC on mitotic chromosomes.
By using a fluorescent protein fused to histone
(His2AvD), the chromosomes can be visualized in
red colour in this still of a video taken from a life
Drosophila embryo. DmOrc2-GFP is shown in green.
DmORC is absent from metaphase chromosomes and
rapidly recruited to the segregating chromosomes in
late anaphase
(A) Nuclei in different stages of the cell cycle are
marked: interphase (black arrow), metaphase
(white arrow), late anaphase (white arrowheads),
telophase (black arrowheads). Scale bar is 20µm.
(B) Enlarged picture of a nucleus moving into metaphase.
(C) Enlarged picture of a nucleus moving out
of metaphase. (for (B),(C): scale bar 5µm, elapsed
time from first picture is indicated.
gate the effects of siRNA mediated knockdown of the
expression of such genes, which results in the inhibition of
DNA synthesis as well as the stalling of cells in mitosis. We
are currently evaluating the use of this technology for the
control of cell proliferation in animal models.
Promoter crosstalk and epigenetic regulation of
transgenes
Mathias Hampf
Transcription units randomly integrated in the chromosomes
of mammalian cells are subject to both epigenetic
control and the influence of nearby transcription signals.
These findings have important implications for the design of
gene expression vectors for transgenesis and gene therapeutic
approaches. It is often desirable to transfer multiple
transcription unit in one step. We are analyzing the effects
these transgenes exert on each other by using an inducible
transcription system. Upon induction of a target gene, a
neighboring “constitutive” transcription unit can be co-regulated
depending on the nature of the promoters used. Vice
versa, these promoters can have a dominant influence over
the characteristics of the inducible transcription unit. To
understand the mechanisms of this crosstalk we need to
control epigenetic effects on transgene expression, like DNA
methylation and chromatin compaction. We recently established
protocols allowing for the reproducible generation of
high level expressing stable cell lines that can escape epigenetic
downregulation over prolonged periods of time. These
cells also show uniform expression when analyzed on the
single cell level. In combination with site-specific recombination
protocols and chromosomal engineering this
approach should allow us to design predictable transgene
expression protocols inert to epigenitic distortions and
might also permit gaining novel insights into the principles
governing the expression of endogenous genes.
Selected Publications
Gossen, M. (2006). Intelligent designs. Gene Ther 13,
1251-1252.
Hampf, M. and Gossen, M. (2006). A protocol for combined
Photinus and Renilla luciferase quantification compatible with
protein assays. Anal Biochem 356, 94-99.
Hampf, M. and Gossen, M. (2007). Promoter crosstalk effects on
gene expression. J Mol Biol 365, 911-920.
Liu, W, Xiong, Y, and Gossen, M. (2006). Stability and homogeneity
of transgene expression in isogenic cells. J Mol Med 84,
57-64.
Ranjan, A, and Gossen, M. (2006). A structural role for ATP in
the formation and stability of the human origin recognition
complex. Proc Natl Acad Sci U S A 103, 4864-4869.
102 Cancer Research