Fig. 1. Generation of a Grem1 loss-of function mutation by gene targeting. (A) The Gre^ORF loss-of-function allele was generated by homologous recombination in ES cells. The entire ORF encoded by exon 2 (e2) was replaced with an IRES-lacZ gene and the Neo^R cassette (flanked by loxP sites indicated by black triangles). Exon 1 (e1) is non-coding and located 8.5 kb upstream of exon 2 (UCSC). The 5' and 3' genomic probes used to screen ES-cell clones by Southern blotting are indicated by black and white boxes, respectively. Thin black lines indicate the sizes of the expected genomic bands detected by these probes. Arrowheads indicate the primers used to detect both wild-type (Wt) and mutant (Gre^ORF) alleles. The relevant restriction enzyme sites are indicated as follows: B, BamHI; E, EcoRV; N, NsiI; Nd, NdeI; X, XbaI. (B,C) Analysis of wild-type (+/+) and correctly targeted heterozygous (+/–) ES-cell clones by Southern blotting using 5' and 3' genomic probes. (D) PCR genotyping of embryos of F2 littermate embryos. (E) Whole-mount in situ hybridization using Gre^ORF/+ embryos at embryonic day 11.0 reveals the identical distribution of Grem1 and lacZ transcripts.