FIGURE 9-10 The human SALL1 enhancer activates expression of a reporter gene in limb buds of the developing mouse embryo. (a) Graphic representation of the conservation of DNA sequence in a region of the human genome (in the interval of chromosome 16 from 50214 kb to 50220.5 kb) about 500 kb downstream from the SALL1 gene, which encodes a zinc-finger transcription repressor. A region of roughly 500 bp of nonprotein-coding sequence is conserved from zebrafish to human. Nine hundred base pairs of human DNA including this conserved region were inserted into a plasmid next to the coding region for E. coli β-galactosidase. (b) The plasmid was microinjected into a pronucleus of a fertilized mouse egg and implanted in the uterus of a pseudopregnant mouse to generate a transgenic mouse embryo with the reporter-gene-containing plasmid incorporated into its genome (see Figure 5-43). (c) After 11.5 days of development, at the time when limb buds develop, the fixed and permeabilized embryo was incubated in X-gal, which is converted by β-galactosidase into an insoluble, intensely blue compound. The results showed that the conserved region contains an enhancer that stimulates strong transcription of the β-galactosidase reporter gene specifically in limb buds.

[Part (a) data from A. Visel et al., 2007. VISTA Enhancer Browser—a database of tissue-specific human enhancers. Nucleic Acids Res. 35:D88–92. Part (b) ©Deco/Alamy. Part (c) republished with permission of Nature, from Pennacchio, L.A., et al., “In vivo enhancer analysis of human conserved non-coding sequences”, Nature, 444, 499–506, 2006; permission conveyed through Copyright Clearance Center, Inc.]