Extended Data Fig. 2: Generation and characterization of mice with transplanted enhancers.

(a) Schematic overview of enhancer replacement strategy. A 4.5 kb mouse genomic region containing the ZRS enhancer is shown together with the vertebrate phyloP conservation (dark blue). The donor vector contained two homology arms with vector-specific sequences for genotyping (green) and a corresponding replaced region containing the transplanted enhancer and mutagenized sgRNA recognition site (black, 5’-agtaccatgcgtgtgtTtTagCC-3’). PCR primers used for genotyping are shown as arrows. See Methods and Kvon et al.⁴⁰ for more details. (b) Shown are the results of PCR genotyping for each knock-in mouse line. One representative sample is shown for each genotype consistent with the results seen for all embryos and mice of the lines represented that were analysed in this study. For gel source data, see Supplementary Fig. 1. (c) Shh mRNA whole mount in situ hybridization analysis in wild type and knock-in mouse embryos (first two columns). The corresponding hind limb skeletal preparations of E18.5 wild type and knock-in mouse embryos are shown (third column). The number of embryos that exhibited representative limb phenotype over the total number of embryos with the genotype is indicated. Genotypes for each row are displayed on the left. (d) E18.5 skeletal staining showing the forelimb (left panel) and hindlimb (right panel) phenotype in heterozygous knock-in embryos.