Figure 4

Functional characterization of linked multi-reporter gene construct in transgenic mice. (A) Imaging of fluorescent reporters in six week old spine under fixed exposure demonstrating intensity differences among different lines. Ibsp-Topaz and Dmp1-mCherry fluorescent reporter genes are easily detected in most lines, however Trap-ECFP in most lines appears highly mosaic expression with only a random cell having ECFP fluorescence. Line 28-2-2 is the only line that has uniform Trap-ECFP reporter expression, but it has no reporter expression for Dmp1 and low expression for Ibsp-Topaz (over exposed inset). (B&C) Confirming the fidelity of the Trap-ECFP reporter. (B) TRAP histological stain on a 3 week old femur that was initially imaged (C) for Trap-ECFP expression. ECFP expression and TRAP staining are essentially identical. (D-E) Genotyping of litters from three separate mouse lines for all three reporter genes. Individual reporters co-segregate together verifying common chromosomal integration. For lines 26-1-5 and 28-4-5 all three reporters are detected, however, in line 28-2-2 DMP1-mCherry reporter is not detected by PCR (D) or viewing of tail biopsies (E). (F) Estimated copy number of DNA construct in different transgenic lines using primers directed against the TRAP reporter. (G) High magnification expression in Ibsp-Topaz and Dmp1-mCherry in trabecular bone of a 6 week old female femur from line 28-4-3.