Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth
Figure 1
Generation of Col2a1-ccn2 transgenic mice.
(A) Schematic representation of the construct of the expression of HA-tagged CCN2 and IRES-LacZ in chondrocytes driven by the 6-kb Col2a1 promoter-enhancer. The original initiation codon of Col2a1 was mutated to CTG to facilitate translation from downstream cDNA. (B) Genotyping of transgenic mice (tg) by PCR to detect the transgene. wt, wild type. The location of the primers used are indicated in “A” by arrows. (C) Skeletal preparation of a newborn mouse after whole-mount X-gal staining, showing cartilage-specific expression of the transgene. (D) Sagittal sections of ulnae from wt and tg after whole-mount X-gal staining. All of the cartilaginous cells showed X-gal staining. The sections were counter-stained with Safranin-O. (E) Analysis of transgene expression by Northern hybridization using total RNA from tg and wt cartilage. LacZ (top) and ccn2 (middle) probes were used to detect transgenic and endogenous ccn2, respectively. (F) Western blot (WB) analysis using cell lysates from tg and wt cartilage and anti-HA antibody recognizing only the CCN2-HA transgene products (left blot). The HA-tagged CCN2 was expressed in cartilage of tg mice. A Western blot of the same cell lysate reacted with anti-actin antibody as a loading control is also shown (right blot).