Figure 1.

Gene replacement for Runx2ΔC. (A) Generation of the mutant locus. The schematic at the top shows the functional domains of Runx proteins and Runx2 exon organization. Domains in the N terminus (N) conserved among Runx transcription factors are indicated: the runt homology/DNA binding domain (RHD) and the nuclear localization signal (NLS). The letters Q and A designate homopolymeric stretches of glutamine and alanine residues unique to Runx2. The C terminus (C) defined by exon 8 includes the nuclear matrix targeting signal (NMTS). Below this schematic is indicated the sequence of the WT (wt) and mutated (mt) allele, and ** denotes the premature stop codon. The bottom portion shows a diagram of target constructs used for introduction of the mutation at the start of exon 8. The C-terminal genomic organization of the WT and mutated alleles are also illustrated. The restriction sites (B, BamHI; E, EcoRI) in the Runx2 genomic locus and the regions of homologous recombination are indicated. Removal of the neo-thymidine kinase cassette by Cre recombinase results in the mutant allele. The loxP sites are shown by filled triangles. The Runx2 gene encodes two major isoforms with two distinct N termini (p56/Type I and p57/Type II) that are generated from two different promoters. Our strategy produces a C-terminal deletion in both isoforms. UTR, untranslated region. (B) Genotyping by PCR analysis of mouse tail genomic DNA from WT (+/+), heterozygous (+/ΔC), and homozygous mutant (ΔC/ΔC) mice. Locations of the forward and reverse primers (which span the lox P site) used as probes for genotyping are indicated by arrows. The presence of the lox P site generates a slower migrating PCR band that represents the Runx2ΔC mutant allele carrying the premature stop codon. (C) Sequence analysis of the PCR products amplified from genomic DNA of WT (+/+) and homozygous mutant (ΔC/ΔC) mice confirms in vivo incorporation of the premature stop codon (denoted by *).