Journal: bioRxiv

Article Title: Functional impact of pathogenic Runt domain mutations in Runx2 in vivo : Insights into the skeletal and dental anomalies of cleidocranial dysplasia

doi: 10.1101/2025.06.18.660258

Figure Lengend Snippet: Generation of Runx2 mutant mice using Platinum TALENs. a Schematic representation of the mouse Runx2 gene with eight exons (labeled E1-E8 below), distal (P1) and proximal (P2) promoters for Type II (MASNSL) and Type I (MRIVD) isoforms, and target sites of Platinum TALENs ( mRunx2-TALEN-L and -R ). A 10 kb scale bar is shown. The SmaI site (red box) includes G695 (underlined). The ssODN contains target sites (green), a T>G silent mutation at 690 creating an ApaI site (blue box), and a G>A mutation at 695 disrupting the SmaI site. b Domain structure of the mRunx2 Type II protein (528 aa) and its corresponding exons. The protein comprises a QA repeat domain, RHD, NLS, PST-rich domain, NMTS, and a C-terminal VWRPY motif. Amino acid positions are indicated above, and the corresponding exons (E1-E8) are shown below. c Nucleotide sequences are shown for the wild-type ( Wt , 670-725), missense mutant ( m , c.695G>A; p.R232Q, 670-725), and two-base deletion ( Δ2 , c.697_698delGA; p.E233TfsTer9, 670-723) alleles. Wt is on top, m in the middle, and Δ2 at the bottom. d Representative electropherograms of the targeted Runx2 region in Runx2 +/+ , Runx2 m/+ , and Runx2 Δ2/+ mice. A black arrow marks T at 690 in Runx2 +/+ and Runx2 m/+ mice. A blue arrow indicates overlapping T and A peaks at 690 in Runx2 m/+ mice, and a red arrow shows overlapping A and G peaks at position 695 in Runx2 m/+ mice.

Article Snippet: TALEN plasmids targeting mouse Runx2 were constructed using the Platinum Gate TALEN Kit (Addgene, Watertown, MA) as previously described .

Techniques: Mutagenesis, TALENs, Labeling