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

Abstract

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.

Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression

“…Bacterial artificial chromosome (BAC) vectors were modified using homologous recombination in E. coli essentially as described (Mortlock et al 2003) to contain a lacZ:Neo (β-geo) fusion cassette into the Bmp2 or Bmp4 transcription unit. Briefly, mouse Bmp2 BACs RP23-85O11 (239,101 kb) and RP23-409L24 (209,640 kb) were modified as previously described (Chandler et al 2007).…”

“…They are also thought to be more resistant to position effects than smaller transgenes (Giraldo and Montoliu 2001;Gong et al 2003). For these reasons, they are particularly useful for studying long-range cis-regulatory phenomena (Mortlock et al 2003;Chandler et al 2007) and for experiments where precise transgene expression is critical, such as cre-recombinase drivers (Lee et al 2001;Copeland et al 2001). In addition, BACs are increasingly used for rescue experiments or overexpression studies.…”

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Because all members of the deletion library contain identical changes at the loxP or lox511 end, comparisons of expression patterns between any two enhancer-trap BACs from the same deletion series are more meaningful as the modifications in each remain constant. Thus BACs can be "scanned" with enhancer-traps to identify distal regulatory elements; and this approach is likely to be less biased than other methods where mutations are targeted to specific sites in the BAC because there is no need to select sequences to test their regulatory potential [30][31][32]34]. In situations where regulatory function is conserved without sequence similarity [10][11][12][13][14], choosing the correct sequence to test might be very difficult as many of the players involved in fine tuning the regulation of gene expression remain unknown.…”

“…Thus BACs of ~200 kb size and containing the β-globin gene were functionalized with EGFP reporter gene using RedET-recombination and expressed in stable erythropoietic cell lines [29]. RecA mediated homologous recombination was used to introduce a LacZ reporter gene cassette into the Gdf6 transcription unit and long-range regulatory sequences mapped using a series of BAC deletions in transgenic mice [30]. Using RecA mediated homologous recombination, 97 mixed CAA-CAG repeats were introduced into the human htt gene in a BAC to build a mouse model of huntingtin's disease [33].…”

“…It has the advantage of using shorter length homologous sequences for recombination to introduce exogenous DNA cassettes into BACs [23]. Both methods have been widely used to engineer a variety of alterations in BAC DNA, such as introducing reporter gene cassettes in frame into the first exon of the target gene, mutating sequences at a target site, and introducing loxP sites [24][25][26][27][28][29][30][31][32][33][34]. Thus BACs of ~200 kb size and containing the β-globin gene were functionalized with EGFP reporter gene using RedET-recombination and expressed in stable erythropoietic cell lines [29].…”