MCB Accepts, published online ahead of print on 15 October 2007

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Mol. Cell. Biol. doi:10.1128/MCB.00498-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Post-Integrative Gene Silencing Within the Sleeping Beauty Transposition System

Brian S. Garrison, Stephen R. Yant, Jacob Giehm Mikkelsen, and Mark A. Kay^*

Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA; Department of Microbiology and Immunology, Stanford University School of Medicine, CA

^* To whom correspondence should be addressed. Email: markay{at}stanford.edu.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel non-selective FACS-based method and discovered that SB integrates 20X-more frequently than previously reported within systems that were dependent on transgene expression and likely subject to post-integrative gene silencing. Over-time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional post-integrative gene silencing which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2'-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multi-component post-integrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.