Fig. 2

Establishing a hypoxia fate-mapping triple-transgenic mouse model. a Design of a triple-transgenic mouse generated by (1) developing a transgenic mouse that produces Cre recombinase in cells exposed to hypoxia followed by (2) breeding to the mT/mG reporter mouse (Jackson Labs; 007676). The double-transgenic mouse (2 T) was then crossed to a (3) mouse expressing the MMTV promoter-driven PyMT oncogene that develops spontaneous breast tumors (3 T). b TLA sequence coverage across the mouse genome by using primer sets 1 and 2 (Supplementary Table 2) was conducted to determine the location of our transgene. The 4xHRE-CRE-ODD transgene integrated at chromosome 3 position 61,062,240. c Triple-transgenic mice were sacrificed at different time points over a 4-month period in order to detect hypoxic cells during breast cancer progression from hyperplasia, to ductal carcinoma in situ (DCIS), to early carcinoma and invasive late-stage carcinoma. H&E-stained sections of paraffin-embedded tissue (left) or fluorescent imaging of frozen tissue sections (right). Marked insets are displayed on the right. The second inset at the DCIS stage highlights early detection of hypoxia