Fig 2: Cre expression indicated by LacZ reporter gene expression in E13.5 mouse embryos resemble the endogenous expression pattern of integrin a11.Cryosections of Cre-positive (ITGA11-Cre+;R26R) embryos were analyzed for ß-galactosidase activity. Strong X-gal staining was noted in the periosteum and cartilage primordium of vertebrae (v), digit of limb (dl), ribs (ri), hip bone, hyoid bone (hb), and heart (h), lung (lu) and Meckel's cartilage (mc) in agreement with expression of endogenous a11 expression at these sites (B). No X-gal staining was visible in the Cre negative (ITGA11-Cre-;R26R) embryos (A). X-gal staining and immunostaining of integrin a11 were performed on sagittal embryo cryosections (C–H). LacZ and integrin a11 (arrows) are expressed in the mesenchymal condensation/periosteum of vertebrae (v) and intervertebral disc (C, D), mesenchyme in forming digits (E, F), periosteum of scapula (sp) (G, H). Scale bars represent 2.5 mm in A and B, 100 µm in C to H.

Fig 3: Cre expression in E16.5 mouse embryos.Embryos were harvested from ITGA11-Cre+;R26R and ITGA11-Cre-;R26R mice at E16.5. ß-galactosidase activity (A) and endogenous expression of integrin a11 (B) were analyzed in embryo cryosections. X-gal positive staining similar as Fig. 2B was observed in the ITGA11-Cre+;R26R embryo (A), scale bar = 5 mm.

Fig 4: Schematic illustration of site-specific insertion of the ITGA11-Cre transgene into the H11P3 locus in mouse genome. C57BL/6 mice homozygous for the modified H11 locus served as embryo donors. The plasmid where the transgene ITGA11-GFPCre is flanked by two attB sites was mixed with in vitro transcribed ϕC31 mRNA and injected into a single pronucleus of each zygote. Insertion of the transgene at the attP site was mediated by ϕC31. Integration of the plasmid bacterial backbone (BB) was not a planned outcome, but according to expression analyses seemed to have little effect on transgene expression.

Fig 5: Low expression of ITGA11-Cre in adult mouse tissues.Western blot analysis showing the total protein expression of ß galactosidase and integrin a11 in the different adult tissues harvested from ITGA11-Cre+;R26R mice. MEFs from transgenic and wild-type mice, respectively, were used as positive and negative controls. GAPDH and Ponceau S stained total proteins were used as loading controls.