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Published online before print August 12, 2003
Genome Research, DOI: 10.1101/gr.1306003
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Letter

A General Approach for Identifying Distant Regulatory Elements Applied to the Gdf6 Gene

Douglas P. Mortlock1, Catherine Guenther and David M. Kingsley2

Department of Developmental Biology and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305-5329, USA

Regulatory sequences in higher genomes can map large distances from gene coding regions, and cannot yet be identified by simple inspection of primary DNA sequence information. Here we describe an efficient method of surveying large genomic regions for gene regulatory information, and subdividing complex sets of distant regulatory elements into smaller intervals for detailed study. The mouse Gdf6 gene is expressed in a number of distinct embryonic locations that are involved in the patterning of skeletal and soft tissues. To identify sequences responsible for Gdf6 regulation, we first isolated a series of overlapping bacterial artificial chromosomes (BACs) that extend varying distances upstream and downstream of the gene. A LacZ reporter cassette was integrated into the Gdf6 transcription unit of each BAC using homologous recombination in bacteria. Each modified BAC was injected into fertilized mouse eggs, and founder transgenic embryos were analyzed for LacZ expression mid-gestation. The overlapping segments defined by the BAC clones revealed five separate regulatory regions that drive LacZ expression in 11 distinct anatomical locations. To further localize sequences that control expression in developing skeletal joints, we created a series of BAC constructs with precise deletions across a putative joint-control region. This approach further narrowed the critical control region to an area containing several stretches of sequence that are highly conserved between mice and humans. A distant 2.9-kilobase fragment containing the highly conserved regions is able to direct very specific expression of a minimal promoter/LacZ reporter in proximal limb joints. These results demonstrate that even distant, complex regulatory sequences can be identified using a combination of BAC scanning, BAC deletion, and comparative sequencing approaches.

The sequence data from this study have been submitted to GenBank under accession no. AC058786. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: E.C. Lee, N.G. Copeland, and A.F. Parlow.]

1 Present address: Program in Human Genetics, Vanderbilt University Medical Center, Nashville, TN 37232-0700, USA.

2 Corresponding author.
E-MAIL kingsley{at}cmgm.Stanford.edu; FAX (650) 725-7739.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1306003. Article published online before print in August 2003.


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