Generation of a novel mouse strain with fibroblast-specific expression of Cre recombinase

Abstract

Cell-specific expression of genes offers the possibility to use their promoters to drive expression of Cre-recombinase, thereby allowing for detailed expression analysis using reporter gene systems, cell lineage tracing, conditional gene deletion, and cell ablation. In this context, current data suggest that the integrin α11 subunit has the potential to serve as a fibroblast biomarker in tissue regeneration and pathology, in particular in wound healing and in tissue- and tumor fibrosis. The mesenchyme-restricted expression pattern of integrin α11 thus prompted us to generate a novel ITGA11-driver Cre mouse strain using a ϕC31 integrase-mediated knock-in approach. In this transgenic mouse, the Cre recombinase is driven by regulatory promoter elements within the 3 kb segment of the human ITGA11 gene. β-Galactosidase staining of embryonic tissues obtained from a transgenic ITGA11-Cre mouse line crossed with Rosa 26R reporter mice (ITGA11-Cre;R26R) revealed ITGA11-driven Cre expression and activity in mesenchymal cells in a variety of mesenchymal tissues in a pattern reminiscent of endogenous α11 protein expression in mouse embryos. Interestingly, X-gal staining of mouse embryonic fibroblasts (MEFs) isolated from the ITGA11-Cre;R26R mice indicated heterogeneity in the MEF population. ITGA11-driven Cre activity was shown in approximately 60% of the MEFs, suggesting that the expression of integrin α11 could be exploited for isolation of different fibroblast populations. ITGA11-driven Cre expression was found to be low in adult mouse tissues but was induced in granulation tissue of excisional wounds and in fibrotic hearts following aortic banding. We predict that the ITGA11-Cre transgenic mouse strain described in this report will be a useful tool in matrix research for the deletion of genes in subsets of fibroblasts in the developing mouse and for determining the function of subsets of pro-fibrotic fibroblasts in tissue fibrosis and in different subsets of cancer-associated fibroblasts in the tumor microenvironment.

Keywords: Cre-recombinase; Fibroblast; Fibroblast-specific; Integrase; Integrin alpha11.

Fig. 1

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. 2

Cre expression indicated by LacZ reporter gene expression in E13.5 mouse embryos resemble the endogenous expression pattern of integrin α11. Cryosections of Cre-positive (ITGA11-Cre+;R26R) embryos were analyzed for β-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 α11 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 α11 were performed on sagittal embryo cryosections (C–H). LacZ and integrin α11 (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. β-galactosidase activity (A) and endogenous expression of integrin α11 (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

Cre is functional in MEFs isolated from ITGA11-Cre;R26R mouse embryos. Western blot showing the endogenous integrin α11 expression level and the expression of β-galactosidase to indicate the Cre activity in the MEF isolates from E13.5 embryos of the transgenic mouse (A). Representative vimentin staining (B) and X-gal staining (C) of the MEFs isolates from either Cre+ or Cre− embryos. Quantification of the MEFs X-gal staining by cell number (D). Cre+ and Cre− MEFs were counted and quantified from ten staining fields of each MEF isolates (five fields from each of the duplex coverslips). The percentage of X-gal-positive cells in each field was summarized in (E). Note absence of β-galactosidase band (A) or X-gal staining (D) in Cre-negative MEFs. Scale bar = 20 μm.

Fig. 5

Low expression of ITGA11-Cre in adult mouse tissues. Western blot analysis showing the total protein expression of β galactosidase and integrin α11 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.

Fig. 6

Induction of ITGA11-Cre in fibrotic hearts. Aortic banding was performed on 8 weeks old ITGA11-Cre+;R26R mice in a protocol resulting in fibrosis. Sham-operated mice were used as controls. (A) The total protein expression of β galactosidase and integrin α11 in both sham- and aortic-banded hearts were analyzed by western blotting. Adjacent heart cryosections were stained with Sirius red (F, G) and X-gal (H, I) from fibrotic regions of aortic banded hearts and the ventricular region of sham-operated heart. In addition, adjacent cryosections were immune-stained with integrin α11 (B, C) and vimentin (D, E), respectively from fibrotic regions of aortic banded hearts and the ventricular region of sham-operated heart. Induction of Cre-activity was observed in the fibrotic area of aortic banded hearts compared to sham-operated heart. Scale bar = 100 μm.

Fig. 7

Induction of ITGA11-Cre in the granulation tissue of healing skin wounds. Two circular wounds of 6 mm diameter were inflicted on the backs of ITGA11-Cre+; R26R and ITGA11-Cre-; R26R mice and harvested on day 7 post injury. (A) Macroscopic aspect of wounds taken immediately following wounding (day 0) and of contracted wounds with scab at day 7. (B) Western blot showing protein levels of β-galactosidase and integrin α11 in lysates of day7 wounds. MEF lysates from Cre-positive mice were used as positive control. β-galactosidase activity was detected in ITGA11-Cre+; R26R but not in Cre-negative wounds. (C–F) Low magnification overview of wounds at day7 is shown, boxes indicate areas shown at higher magnification in (G, H). (I) Consecutive wound sections were immunostained with antibodies to αSMA to visualize wound myofibroblasts (I). Scale bars represent 500 μm in (C, F), 250 μm in (D, E) and 100 μm in (G–I).