Targeting Silence: The Use of Site-Specific Recombination to Introduce In Vitro Methylated DNA into the Genome
Dirk Schübeler,
Matthew C. Lorincz , and
Mark Groudine
The authors are in the Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA. E-mail: dschubel{at}fhcrc.org. M. Groudine is also in the Department of Radiation Oncology, University of Washington Medical School, Seattle, WA 98195, USA.
Abstract:
DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.
