Gli1 cre ert2 mice

C57BL/6 mice expressing H3.1-iCOUNT were generated by PolyGene (Switzerland). C57BL/6-derived ESCs were transfected with the iCOUNT cassette targeting H3.1 as described above and selected using G418. Single clones were picked, expanded and correct insertion was verified by PCR. Positive cells were injected into blastocysts and 6 chimeras were born. Crossing three chimeras to gray C57BL/6 females yielded heterozygous H3.1-iCOUNT positive animals. To generate homozygous H3.1-iCOUNT mice, H3.1-iCOUNT ± mice were crossed with each other. Alternatively, H3.1-iCOUNT animals were crossed to Gli1:Cre-ER^T2 mice (Jackson Lab No 007913: (Ahn and Joyner, 2004 (link)) and ROSA26:Cre-ER^T2 mice (Jackson Lab No 008463; (Ventura et al., 2007 (link)). Animals were always genotyped to distinguish WT, heterozygous and homozygous H3.1-iCOUNT animals (see PCR above). H3.1 tagging caused mosaic expression of the RITE cassette, as there 9 different genes encoding for the histone-variants H3.1 and H3.2. within the HIST1 gene cluster on chromosome 13 (Wang et al., 1996 (link)). Indeed, we observed mosaic expression and mice that had the RITE cassette correctly inserted (verified via genomic PCR) but did not express the transgene.

Gli1‐CreER^T2 mice, Rosa26‐tdTomato^flox/flox mice and Tgfbr2^flox/flox mice were obtained from Jackson Laboratory. For lineage‐tracing experiments, a double heterozygous Gli1‐CreER^T2;Rosa26‐tdTomato^flox/wt (Tomato^Gli1ER) mice were generated, and tamoxifen (1 mg/10 g body weight/day, diluted in corn oil) was injected intraperitoneally into 1‐month‐old mice for 3 consecutive days. To investigate the role of TGF‐β signalling in Gli1⁺ periosteal cells in fracture healing, Gli1‐CreER^T2;Tgfbr2^flox/flox (Tgfbr2^Gli1ER) mice and Gli1‐CreER^T2;Tgfbr2^flox/flox; Rosa26‐tdTomato^flox/wt (Tgfbr2^Gli1ER;ROSA^tdTomato) mice were generated following with 3 consecutive intraperitoneal injections of tamoxifen at 1 month of age, or mice were subcutaneously injected with TGF‐β neutralizing antibody (5 mg/kg body weight) at the fracture site once every 2 days starting immediately after fracture. The specific information of transgenic mice were provide in Table 1. Both male and female mice were used in lineage‐tracing studies, but only males were subjected to fracture surgery to avoid sex‐dependent difference. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (LZ12H27001).

Male C57BL/6 mice were purchased from Sun Yat-sen University, Guangzhou, China, and male Gli1-Cre^ERT2 mice and R26^tdTomato mice were purchased from the Jackson Laboratory, USA. We used age-matched 8-week-old male mice from the same background in all animal experiments. Animals were grouped using a method of randomization. No sample size estimation was performed. All these experiments were performed under an institutionally approved protocol (Sun Yat-sen University, SYSU-IACUC-2021-000692).