Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes - PubMed (original) (raw)

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

R J DiLeone et al. Proc Natl Acad Sci U S A. 2000.

Abstract

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

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Figures

Figure 1

Figure 1

BAC coinjections drive expression of a lacZ reporter gene in previously detected Bmp5 expression domains. (A) The position of BAC 551 relative to the Bmp5 gene. Dotted lines at the end of the insert bracket minimal and maximal extent of the insert based on restriction mapping and probe hybridization. The arrows on the 3′ side of the Bmp5 exons indicate positions of chromosome breakpoints in two short ear regulatory mutations, se30DThWb and se4CHLd (17). Enhancers previously detected by reporter transgenic work with phage clones (17) are labeled and indicated by dotted lines. (B) Embryos (E14.5–15.0) transgenic for BAC 551 and hsp-lacZ transgenes show reporter gene expression (arrowheads and small arrows) in areas previously detected by phage clone reporter constructs and in the xiphisternum and ureter–bladder (italics), locations also known to express Bmp5 (25). (C) Embryos transgenic for BAC 551 and the Bmp5prom-lacZ transgenes show a very similar overall expression pattern.

Figure 2

Figure 2

Promoter conservation among the mouse, human, and chicken Bmp5 genes. The transcription start site in mice is shown as the +1 arrow at the bottom. The blackened boxes indicate base pairs that are identical in all three species. Previously published Bmp5 sequence (21) begins at base +32 marked with an asterisk.

Figure 3

Figure 3

BACs 199 and 178 drive expression of lacZ at sites predicted from effects of regulatory mutations on endogenous Bmp5 expression. (A) BACs 199 and 178 extend proximal of the se 30DThWb and distal of the se4CHLd breakpoint, respectively. Previous expression studies suggest that control elements for the indicated sites also should be located proximal or distal of these breakpoints. (B) BAC 199 drives expression in three sites predicted by previous studies: the ribs (top arrow), the finger tips (middle arrow), and the intestines (bottom arrow). A frontal view (C) shows that the ribs, but not the sternum, display robust reporter expression, consistent with normal rib expression (arrowhead) and reduced sternum expression (arrow) of Bmp5 in se30DThWb embryos (E and F). In contrast, BAC 178 (L) drives strong expression in the sternal bands as well as some expression in the ribs (K). This is consistent with regulatory mutant data that place the sternal enhancer 3′ of the se30DThWb breakpoint and BAC 551 data that place it further 3′ as shown in A. The intestinal mesenchyme enhancer activity in BAC 199 (B and D) matches Bmp5 expression at the same site (G) and is consistent with normal Bmp5 expression in se30DThWb embryos (H). The BAC 178-driven reporter expression in the base of the ear (arrowheads in L and M) coincides with one part of the endogenous Bmp5 ear expression domain (arrowhead in I). The position of this enhancer in the 3′ BAC 178 is consistent with the effects of se4CHLd on Bmp5 expression at that site (arrowhead in J).

Figure 4

Figure 4

Mice transgenic for BAC 551 and a hsp-BMP5 construct show rescue of short ear phenotypes. Two transgenic short ear mice (se/se;TG) are shown on either side of a nontransgenic short ear littermate (se/se) in A or a se/+ long ear control (wt) in B. The ear length is rescued substantially in transgenic mice, but only to 80–90% of total ear length seen in wild-type mice. (C_–_E) Xiphisternums (arrowheads) from wild-type, se/se;TG, and se/se mice show that the presence of the transgene restores the xiphisternum to wild-type length. Alcian blue- and Alizarin red-stained regions correspond to regions of cartilage and bone, respectively.

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