Figure 1. Generation and expression analysis of SHOX-transgenic mice.

(A): The SHOXa cDNA was tagged with a Lumio and SV40 Poly(A) sequence and cloned under the control of a murine Col2a1 promotor/enhancer expression cassette. (B): Genotyping was performed using specific primers spanning the first 409 nucleotides of the SHOXa cDNA. No PCR product was detected in wildtype animals. (C):-Southern Blot analysis of the two transgenic lines (1 and 2) used for our investigations. Genomic DNA was digested with BamHI, EcoRV and Hind III. BamHI digestion results in a 1.3 kb fragment that corresponds to the Lumio/SV40-tagged SHOX cDNA, which was flanked by BamHI sites. The presence of only one signal per lane indicates a single integration site of the transgene. (D): Relative quantitative expression of Col2a1 and SHOXa transcripts in limbs of wildtype and transgenic littermates (N = 5–8 per litter) at E12.5, E13.5 and E14.5. The expression of the transgene corresponds to the expression dynamics of Col2a1. SHOX levels are generally low with highest expression at E12.5. Values are variable among individual animals as indicated by the standard deviation (SD). (E): WISH of wildtype (Wt) and transgenic (Tg) embryonic limbs from E11.5-E14.5 (N = 20 for each stage). The transgene is weakly expressed in the developing limb at E11.5 and becomes defined around the cartilaginous anlagen at E12.5. From E13.5 onwards, the expression is mainly seen in the mesenchyme around the developing cartilage and in the perichondrium and decreases during later stages.