alexa Minicircle performance depending on S MAR-nuclear matrix interactions.
Molecular Biology

Molecular Biology

Journal of Cell Science & Therapy

Author(s): Broll S, Oumard A, Hahn K, Schambach A, Bode J

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Abstract The ideal vector for cell and tissue modification does not depend on integration but rather behaves as an independent functional unit that replicates as an episome. Based on a scaffold/matrix attachment region (S/MAR), we have introduced, in 2006, an approximately 4-kb replicating nonviral minicircle able to exploit the cellular replication machinery in a way reminiscent of ARS vectors. Consisting of only one active transcription unit and the S/MAR, it resists silencing as it is free of prokaryotic vector parts and drug selection markers. The rate of final establishment in the nuclear architecture is moderate but comparable to Epstein-Barr virus-based episomes (<5\%). Here, we demonstrate that this parameter can be improved if the host cell chromatin is opened by histone hyperacetylation prior to transfection. It remains unaffected, however, by cell cycle position. Still, this class of episomes revealed intrinsic instability and integration after 5 months of continuous culture. In vivo evolution enabled the effective reduction of S/MAR size from 2 kb to 733 bp (resulting in a minicircle of approximately 3 kb) with largely improved stability and cloning capacity. Investigation of individual clones served to prove persistent and homogenous expression, which is ascribed to stable association with nuclear attachment sites. Optimum expression levels were shown to depend on the authentic usage of a polyadenylation site 3' from the S/MAR as anticipated by the stress-induced duplex destabilization algorithm, which finds increasing use to predict the functional parameters of these systems. Copyright 2009 Elsevier Ltd. All rights reserved. This article was published in J Mol Biol and referenced in Journal of Cell Science & Therapy

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