9th Annual Workshop, October 28-31, 1999
Co-sponsored by the U.S. Department of Energy
BODE
Scaffold/matrix attached regions (S/MAR Elements): Detection and activities in vivo
Jürgen Bode, Alexandra Baer, Angela Knopp, Dirk Schübeler, Jost Seibler, Craig Benham Armin Baiker , and Hans-Joachim Lipps
GBF - National Institute for Biotechnological Research, Braunschweig, Germany
S/MARs have been discovered more than a decade ago and have been defined as DNA-elements staying attached to or associating with the nuclear skeleton after the extraction of the histones and soluble factors from eukaryotic nuclei. The macroscopic binding properties have been carefully characterized and are now mainly ascribed to the lamins as well as scaffold attachment factor A (SAF-A = hnRNP-U). While S/MARs do not conform to any obvious sequence consensus, their recognition appears to be governed by structural features, most significantly their propensity to expose single strands under negative superhelical tension. This property has been used successfully to localize S/MARs in SIDD (stress-induced duplex destabilization) profiles, to predict their activity and and to guide the design of artificial S/MARs (Review: Gene Therapy Mol. Biol. 1, 551, 1998).
The first generally accepted biological activity of S/MARs was the augmentation of transcription initiation rates which operates by a mechanism distinct from enhancement. Since then the number of potential biological functions has increased and can be classified as follows:
This overlap of functions has made difficult the unambiguous demonstration of any of these components. Standard transfection techniques cannot, for instance, discriminate between a targeting action of a S/MARs and a cis-activity of the same element. It was originally for this reason that we have developed a variety of techniques based on site-specific recombination systems like Flp/FRT and Cre/loxP. With these techniques, complete chromatin domains can be decomposed, inverted or elaborated at a predefined chromosomal locus - a concept with obvious relevance for the rational construction of cell lines with a high and consistent expression and the efficient generation of transgenic animals with a predictable regulation of the transgene.. Our most advanced system, the recombinase-mediated cassette exchange (RMCE) permits the exchange of an integrated cassette which is flanked by an FRT-site and a FRT-mutant. During RMCE, any pre-existing positive/negative marker is removed resulting in an integration event that is not perturbed by either a co-expressed selection marker or prokaryotic vector sequences. It will allow the efficient stepwise, stable (but reversible) introduction of insulator/bordering elements to support the autonomous expression of a transgene at a given genomic site.
So far the stable modification of target cells is mostly achieved by integrating vectors although their expression is rapidly silenced and may give raise to insertional mutagenesis or recombination. While episomal vectors exist, the function of their replication origins usually relies on virally encoded transacting factors which often lead to cellular transformation. Major recent efforts have therefore been devoted to the use of S/MAR-ori sequences from the human genome to obtain vectors which replicate autonomously and thereby provide a stable and high-level expression. We have demonstrated that the function of the large- T oncogen on the SV40 origin can be substituted for by a human S/MAR element and that this combination of S/MAR and ori sequences efficiently prevents integration. There are indications that a S/MAR directs the episome to replication sites on the nuclear matrix, recruiting all the endogenous cellular factors which are required for its propagation and stable extrachromosomal maintenance.