Figure 1: Lineage-tracing using tamoxifen-inducible multicolor labeling system to track the cell-fate of Bmi1-positive tumorigenic cells in various models of intestinal tumors.
(a) Schematic protocol of the CreERT2-mediatid multicolor labeling using Bmi1CreERT/+; Rosa26rbw/+ mice. Rainbow (rbw) construct is knocked-in into Rosa26 locus and consists of three loxP variants: lox2271, loxN, and loxP, and subsequent cDNA for Green Fluorescent protein (GFP) followed by cDNAs for mCerulean, mOrange and mCherry, which are proceeded by lox2271, loxN, and loxP, respectively. This mouse was crossed with Bmi1CreERT/+ line for the purpose of multicolor lineage tracing of Bmi1-positive cells. When this mouse, all the cells of which express GFP, received tamoxifen, recombination is occurred in Bmi1-positive cells mediated by CreERT2, leading to GFP deletion and random exercise between two lox2271s, loxNs, or loxPs. As a result, mCerulean, mOrange or mCherry moves next to CAG promoter, resulting in random expression of either fluorescent protein. After tumor was confirmed to occur, the mice were injected with tamoxifen to induce multicolor labelling. (b) Schematic protocol of lineage-tracing of Bmi1-positive cells in tumor-bearing FAP model mice. FAP spontaneously occurs in APCmin/+ mice before the age of 140. P, Postnatal day. (f) Schematic protocol of lineage-tracing of Bmi1-positive cells in tumor-bearing two-step carcinogenesis model mice. When APCmin/+ mice, which develop FAP, are administrated with 2% DSS from the age of 28 to 35, tumorigenesis is accelerated to generate colon adenoma. P, Postnatal day. (j) Schematic protocol of lineage-tracing of Bmi1-positive cells in tumor-bearing sporadic carcinogenesis model mice. P, Postnatal day. (c–e,g–i,k–m) Representative traced images of Bmi1+ cell induced in developing tumors. Tumors were harvested at 3 days after induction (c,g and k), 7 days after induction (d,h and l) and 28 days after induction (e,i and m) in FAP model, two-step carcinogenesis model, and sporadic carcinogenesis model, respectively. G; GFP, C; mCerulean, O; mOrange, Ch; mCherry. Scale bars, white; 100 μm, yellow; 50 μm, red; 20 μm, green; 10 μm.
