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J. Virol., 01 1998, 339-348, Vol 72, No. 1
C Laker, J Meyer, A Schopen, J Friel, C Heberlein, W Ostertag and C Stocking
The use of retroviral vectors for gene transfer into animals has been
severely hampered by the lack of provirus transcription in the early embryo
and embryonic stem (ES) cells. This primary block in provirus expression is
maintained in differentiated cells by a cis-acting mechanism that is not
well characterized. Retroviral vectors based on the murine embryonal stem
cell virus (MESV), which overcome the transcriptional block in ES cells,
were constructed to investigate this secondary mechanism. These vectors
transferred G418 resistance to ES cells with the same efficiency as to
fibroblasts, but overall transcript levels were greatly reduced. A mosaic
but stable expression pattern was observed when single cells from
G418-resistant clones were replated in G418 or assayed for expression of
LacZ or interleukin-3. The expression levels in independent clones were
variable and correlated inversely with methylation. However, a second, more
pronounced, block to transcription was found upon differentiation
induction. Differentiation of the infected ES cells to cells permissive for
retroviral expression resulted in repression and complete extinction of
provirus expression. Extinction was not accompanied by increased levels of
methylation. Provirus expression is thus regulated by two independent
cis-acting mechanisms: (i) partial repression in the undifferentiated
state, accompanied by increased methylation but compatible with long-term,
low expression of retroviral genes, and (ii) total repression and
extinction during early stages of differentiation, apparently independent
of changes in methylation. These results indicate a time window early
during the transition from an undifferentiated to a differentiated stage in
which provirus expression is silenced. The mechanisms are presently
unknown, but elucidation of these events will have an important impact on
vector development for targeting stem cells and for gene therapy.
Copyright © 1998, American Society for Microbiology
Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation
Abteilung fur Zell- und Virusgenetik, Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg, Germany.
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