Extended Data Fig. 4: Recombination of wild-type confetti clones with TAT-Cre or tamoxifen results in similar labelling efficiencies.
From: Mechanisms that clear mutations drive field cancerization in mammary tissue
a, Schematic representation of the R26R-Confetti construct (left), which was recombined sporadically through an intraperitoneal injection with a low dose of tamoxifen in the presence of R26-CreERT2 (R26R-Confettihet;R26-CreERT2het mouse model), which is the gold standard method, or through intraductal injection of TAT-Cre recombinant protein. b, Confocal overview image of a whole-mount 4th mammary gland derived from a R26R-Confettihet;R26-CreERT2het adult female mouse 14 days after tracing initiation by tamoxifen-mediated recombination. Zooms show ducts containing single confetti-labelled cells of both basal and luminal origin, representative of the initial labelling density after tracing initiation. Ductal tree is stained with alpha-smooth muscle actin (SMA) depicted in white, which marks the basal cell layer. Scale bar left image represents 1 mm, scale bar right images represents 100 µm. c, Quantification of the confetti-positive cell fraction 14 days after tamoxifen-mediated recombination. Each dot represents the fraction of recombined cells in a randomly selected area within each mammary gland of approximately 1 ×1 mm, n = 6 glands derived from 6 different mice. Basal cells are normalized to the total number of basal cells in the selected area (blue dots) and luminal cells are normalized to the total number of luminal cells in the selected area (cyan dots). Error bar represents mean ± s.d. d, Confocal overview image of a whole-mount 4th mammary gland derived from a R26R-Confettihet adult female mouse, 14 days after tracing initiation recombined by the TAT-Cre intraductal injection method. Zooms show ducts containing single confetti-labelled cells of both basal and luminal origin, representative of the initial labelling density after tracing initiation. Ductal tree is stained with E-cadherin (ECAD) depicted in white, which marks the luminal cell layer. Scale bar left image represents 1 mm, scale bar right images represents 100 µm. e, Quantification of the confetti-positive cell fraction 14 days after TAT-Cre-mediated recombination for basal (blue dots) and luminal (cyan dots) cells. Each dot represents the fraction of recombined cells in a randomly selected area within each mammary gland of approximately 1 ×1 mm, n = 3 glands derived from 3 different mice. Error bar represents mean ± s.d. Note that recombination efficiencies of luminal and basal cell populations are similar between the tamoxifen- and TAT-Cre-induced recombination techniques. Both induction methods result in the recombination of approximately one labelled cell for every 100–200 cells. As the Confetti construct comprises four distinct colours, there is, on average, one cell labeled with a confetti colour per 400–800 cells. Considering that a MaSC-progeny unit consists of approximately 5 to 10 cells, a single confetti-labeled cell is induced in 1 out of 40–80 units. Importantly, over time, many clones become extinct (Fig. 2d), leading to a dilution in the number of clones and making collisions even less likely.
