Runx2-I Isoform Contributes to Fetal Bone Formation Even in the Absence of Specific N-Terminal Amino Acids
Figure 4
The lethality in Runx2-Ineo/neo mice was rescued by neo cassette deletion.
Runx2-Ineo/+ mice crossed with a Cre deleter mice to remove the neo cassette. (A) Schematic illustration of depletion of the neo cassette. Relevant EcoR V (EV) and Sal I (SI) recognition sites are indicated. (B) Deletion of the neo cassette was confirmed by PCR. The primers depicted in panel (A) were used to detect the wild-type allele (WT-Fw and Rv) and the neo-deleted allele (loxP-Fw and Rv). The PCR products were 1.1 kb in size for both the wild-type and the neo-deleted allele. +/+, wild-type; TGA/+, heterozygous; TGA/TGA, homozygous. (C) Southern blot analysis using the genome of the indicated Runx2-I-targeted littermates after digestion by EcoR V and Sal I. The membrane was blotted with the probe shown in panel (A). The size of the band was 7.2 kb in the wild-type allele and 1.9 kb in the neo-deleted allele. (D) Fetal calvarial cells were prepared from generated mice. RNA was purified from mice of each genotype and then analyzed by quantitative real-time PCR for expression levels of total Runx2, Runx2-I, and Runx2-II. Data are representative of three independent experiments and are means ± SEM (n = 3/group).