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 @entity19 @entity15138 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 @entity15138 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 @entity15138 transcription unit of each BAC using homologous recombination in bacteria. Each modified BAC was injected into fertilized @entity19 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 @entity1141 , 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 @entity19 and @entity1 . 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.

A general approach for identifying distant regulatory elements applied to the XXXX gene.

@entity19 :: ('10090', 'Species') :: ['mice', 'mouse'] @entity1 :: ('9606', 'Species') :: ['humans'] @entity1141 :: ('MESH:C535662', 'Disease') :: ['skeletal joints'] @entity15138 :: ('242316(Tax:10090)', 'Gene') :: ['Gdf6']
@entity15138 :: (242316(Tax:10090),Gene) :: ['Gdf6']
