Tracing Intestinal Stem Cell Lineages

Lgr5^Dtr-GFP (MGI:5294798) (Tian et al. 2011 (link)), R26R^Tom (MGI:3813512) (Madisen et al. 2010 (link)), Ngn3^Cre/ERT2 (MGI:5449724) (Li et al. 2012 (link); Li et al. 2019 (link)), and Lgr5-EGFP-IRES-creERT2 (MGI:J127123) (Barker et al. 2007 (link)) mice were maintained on a predominantly C57BL/6 background. Animals were housed under specific pathogen-free conditions in 12-h light/dark cycles at 23°C ± 1°C and 55% ± 15% humidity. Food and water were provided ad libitum. Animals were weaned 21–28 d after birth and handled and euthanized according to procedures approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute. Mice were at least 8 wk old at the time of experiments and cell isolations. Mice of both sexes were used in the experiments, with littermate controls. Ngn3-Cre;Lgr5^Dtr;R26^Tom mice were injected with two daily doses of intraperitoneal tamoxifen (TAM; 50 mg/kg; Sigma T5648), and whole SI was harvested 12, 24, 48, and 72 h after the second dose (Supplemental Fig. S1A). To ablate ISCs, mice received one dose of intraperitoneal diphtheria toxin (DT; 50 µg/kg; Sigma D0564) at the same time as the second TAM injection.