Skip to main content
. 2021 Jul 26;10:e64513. doi: 10.7554/eLife.64513

Figure 2. Col2-Q system: a double-color genetic label-retaining strategy to identify and isolate slow-cycling chondrocytes of the growth plate.

Figure 2.

(a) ‘Col2-Q’ quadruple transgenic system composed of two chondrocyte-specific bigenic Col2-Tet-Off (Col2a1-tTA; TRE-H2B-EGFP) and Col2-iTOM (Col2a1-creER; R26R-tdTomato) systems. H2B-EGFP expression can be shut off by doxycycline diet (2 mg/g diet), while tdTomato expression can be induced by two doses of tamoxifen (4 mg) administered shortly prior to analysis (3 and 2 days before). (b) Experimental design to identify label-retaining chondrocytes (LRCs) in the growth plate. Col2-Q mice are fed with doxycycline (Doxy) starting from postnatal day (P) 21 (Chase). The mice are analyzed after the indicated number of weeks; at each time point, two doses of tamoxifen are administered shortly before analysis to induce tdTomato expression. (c) Diagram for predicted outcomes. LRCs are expected to retain green nuclei with red cytoplasm and located at the resting zone. Non-LRCs have increasingly dilute levels of H2B-GFP as they become proliferative and further differentiated. Rest.: resting zone, Prolif.: proliferating zone, Hyper.: hypertrophic zone. (d,e) Col2-Q distal femur growth plates with tamoxifen injection shortly before analysis. (d): No chase, without Doxy at P21. (e): After 4 weeks of chase, on Doxy for 4 weeks at P49, right panel: high-power confocal image. Arrowhead: label-retaining chondrocytes. AC: articular cartilage, SOC: secondary ossification center, GP: growth plate. Dotted line: border between growth plate and secondary ossification center. Blue: DAPI, gray: DIC. Scale bars: 500 µm, 20 µm (fluorescent scope in e).