Tracing Scleraxis-Expressing Cells in Postnatal Fibrocartilage

All experimental protocols followed ARRIVE (Animal Research Reporting of In vivo Experiments) guidelines and were assessed and approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M College of Dentistry.
To trace Scleraxis-expressing tendon-derived cells during early postnatal fibrocartilage formation, Scx^CreERT2 mice (provided by Dr. Ronen Schweitzer) 43 (link) were crossed to R26R^tdTomato reporter mice (stock number: 009705) and named Scx^Lin. A single intraperitoneal injection of tamoxifen (75mg/kg body weight; T5648, Sigma-Aldrich) dissolved in 90% corn oil (C8267, Sigma-Aldrich) and 10% ethanol 25 (link) was administered at P3. Animals were sacrificed at P4, P9, P19, P33, and P60, separately, corresponding to 24 hours, 1 week, 2 weeks, 4 weeks, and 8 weeks after the tamoxifen induction. To trace the late activated Scleraxis-expressing cells during fibrocartilage formation, tamoxifen was administrated at P28, and the mice were harvested at P35 and P56, respectively, corresponding to 1 week and 4 weeks after the tamoxifen induction.
To trace Col1-expressing fibroblasts-derived cells, 3.2kb Col1^CreERT2 mice 52 (link) were crossed to R26R^tdTomato and named Col1^Lin. Single tamoxifen was administrated at P3, and the forelimbs of mice were harvested at P4, P9, P33, and P60, corresponding to 24 hours, 1 week, 4 weeks, and 8 weeks after the tamoxifen induction.
To generate triple transgenic mice to ablate Scx^Lin cells conditionally, Scx^Lin mice were crossed with R26R^DTA/+ mice (stock number: 009669), with the Scx^Lin as controls. Tamoxifen was administrated once daily for two consecutive days starting from P3, and the forelimbs were harvested at P33.
To constitutively activate β-catenin in Scx^Lin cells (CA-β-cat, Scx^Lin mice were crossed with β-catenin ^{flox(Ex3)/flox(Ex3)} mice 54 (link). Tamoxifen was administrated twice per week, starting from P3 to P33, and then the animals were sacrificed at P60.