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Molecular and Cellular Biology, December 2007, p. 8824-8833, Vol. 27, No. 24
0270-7306/07/$08.00+0     doi:10.1128/MCB.00498-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Postintegrative Gene Silencing within the Sleeping Beauty Transposition System{triangledown}

Brian S. Garrison,1,2,{dagger} Stephen R. Yant,1 Jacob Giehm Mikkelsen,1,{ddagger} and Mark A. Kay1*

Departments of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California,1 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California2

Received 22 March 2007/ Returned for modification 6 June 2007/ Accepted 2 October 2007

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 nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates ~20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative 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 multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.

* Corresponding author. Mailing address: Stanford University School of Medicine, Department of Pediatrics, 300 Pasteur Dr., Room G-305, Stanford, CA 94305-5208. Phone: (650) 498-6531. Fax: (650) 498-6540. E-mail: markay{at}stanford.edu

{triangledown} Published ahead of print on 15 October 2007.

{dagger} Present address: Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA.

{ddagger} Present address: Department of Human Genetics, University of Aarhus, Aarhus, Denmark.

Molecular and Cellular Biology, December 2007, p. 8824-8833, Vol. 27, No. 24
0270-7306/07/$08.00+0     doi:10.1128/MCB.00498-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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