GBF, German Research Center for
Mascheroder Weg 1
presenter: J. Bode
J. Bode1, A. Baer1, E. Ernst1, S. Goetze1,
A. Knopp1, K. Nehlsen1, C. Benham2, H.-J. Lipps3
and A. Baiker4
1GBF - German Research Center for Biotechnology/Epigenetic Regulation, D-38124 Braunschweig; email@example.com
2The Mount Sinai Med. Center, New York, NY 10029 , USA; firstname.lastname@example.org:
3Institut für Zellbiologie, Universität Witten/Herdecke, D-58448 Witten, email@example.com
4Stanford University/Department of Pediatrics, Stanford, CA 94305-5208; firstname.lastname@example.org
The human interferon gene cluster on the short arm of chromosome 9 comprises 26 genes the functional members of which are separated by highly efficient scaffold/matrix attached regions (S/MARs). We have refined in vitro and biomathematical methods to predict the activity of these elements which are characterized by their pronounced propensity to separate strands under superhelical tension. Other features of individual chromatin domains which become apparent in stress-induced duplex-destabilization (SIDD) profiles are the localization of regulatory elements (promoters and DNAse I hypersensitive sites) and of chromosomal breakpoints which explain the genomic instability of this genomic locus. LMPCR techniques have been used to verify the predicted state of DNA at base resolution under superhelical tension and in the living cell.
We have developed a number of in vivo assays to define the spectrum of biological activities for S/MAR elements, among these the augmentation of transcription initiation rates which is distinct from enhancement. S/MARs also interfere with the methylation of transgenes and can be applied to stabilize the long-term expression, particularly of retroviral vectors. Utilizing recombinase-mediated cassette exchange techniques (RMCE) we have initiated a series of experiments to unravel the potential of S/MARs to function as an insulator, a cis-acting or a targeting element. PCR techniques have been applied to identify their role as hotspots of recombination and their participation in deletion and translocation events.
Conventional integrating vectors for eukaryotic cells suffer from a number of limitations which include the possibilities of insertional mutagenesis or silencing of the transgene. So far, only viral vectors derived from SV40-, BPV- or EBV replicate episomally in some eukaryotic cells. The replication origins of these vectors require the support by virally encoded trans-acting factors which can lead to cellular transformation. We present an entirely new vector type in which the function of a viral origin depends of an active S/MAR element. This vector replicates episomally in a number of cell lines and primary cells and is maintained at a constant low copy number in the absence of selection pressure. The functional relevance of matrix association is demonstrated by in situ hybridization and nuclear matrix fractionation procedures.
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