B6 cg gt rosa 26sortm9 cag tdtomato hze j mice

C57BL/6J female mice were from Charles River. The C57BL/6‐Tg(Cdh5‐cre/ERT2)1Rha transgenic mice were kindly provided by Prof. Sarah de Val (University of Oxford), and permission to use them was obtained from Prof. Ralf Adams (Max Planck Institute for Molecular Biomedicine), B6.Cg‐Gt(ROSA)26Sortm9(CAG‐tdTomato)Hze/J mice were purchased from Jackson Laboratory (Stock Number: 007909). Female Ve‐CreERT2 mice and male GtRosa26 reporter mice were crossed to obtain C57BL/6‐Tg(Cdh5‐cre/ERT2)1Rha‐Gt(ROSA)26Sortm9(CAG‐tdTomato)Hze/J mice (VE‐TOM mice) and were bred in our facility. All animal experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 as amended (Amendment Regulations 2012 [SI 2012/3039]), under the authority of a UK Home Office Project License (PPL 30/2922 and PCDCAFDE0), with local ethical approval from the University of Oxford Animal Welfare and Ethical Review Panel. Mice were randomized to control versus treatment groups, and the investigators were not blinded to the experiments. Collection and stopping points were predetermined. Additionally, the experiments were terminated and mice humanely culled if tumors grew up to the size allowed on our animal license or if the implantation of the imaging window failed.

Five- to 6-week-old male C57BL6/J mice, Lgr5-eGFP-IRES-CreERT2 mice, Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)Luo/J mice, and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mice (Jackson laboratories) were maintained ad libitum and all studies were performed under the guidelines and protocols of the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. All the animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center (ACUP number 2016-2316).

All animal experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and were consistent with local, state, and federal regulations as applicable. Mice were housed in a barrier facility with a 12-h light/dark cycle and maintained on standard chow (2916 Teklad Global). C57BL/6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J mice (also known as Ai9 or Ai9(RCL-tdT) mice) were obtained from The Jackson Laboratory (007909) and bred to maintain homozygous expression of the Cre reporter allele that has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent tdTomato protein. Following Cas9/sgRNA RNPs-mediated gene editing, Ai9 mice will express tdTomato fluorescence. Ai9 mice are congenic on the C57BL/6J genetic background. ΔEx44 DMD mice were provided by Prof. Eric Olson’s lab^{44 (link)}.

To investigate the role of Lgr5-expressing intestinal stem cells (ISCs) in tissue regeneration under homeostatic conditions and in response to radiation injury, an in vivo lineage tracing assay was performed. Lgr5-eGFP-IRES-CreERT2 mice were crossed with B6. Cg-Gt (ROSA)26Sortm9(CAG-tdTomato) Hze/J mice (Jackson Laboratories) to generate the Lgr5-eGFP- IRES-CreERT2; Rosa26-CAG-tdTomato heterozygote mice. To initiate lineage tracing, adult Cre reporter mice were injected with tamoxifen (Sigma-Aldrich, St. Louis, MO, USA; 9 mg per 40 g body weight, intraperitoneally), allowing for the labeling of Lgr5+ lineages and their subsequent tdTomato (tdT)-positive progeny. This approach enabled the identification and tracking of Lgr5 ISC lineages. For radiation injury studies, mice received 12.5 Gy of AIR, and tissue was harvested on day 10 post irradiation.

Eight- to twelve-week-old male and female C57BL6/J mice, Lgr5-eGFP-IRES-CreERT2 mice, Gt (ROSA)26Sortm4(ACTB-tdTomato- EGFP) Luo/J mice, and B6. Cg-Gt (ROSA)26Sortm9(CAG- tdTomato) Hze/J mice were purchased from Jackson laboratories. Lgr5-eGFP-IRES-CreERT2 mice were crossed with Gt (ROSA)26Sortm4(ACTB-tdTomato-EGFP) Luo/J mice (Jackson Laboratories) to generate Lgr5/eGFP-IRES-Cre-ERT2; R26-ACTB-tdTomato-EGFP [23 (link),24 (link)]. In Gt (ROSA)26Sortm4(ACTB- tdTomato-EGFP) Luo/J mice tdTomato is constitutively expressed (independent of Cre recombination) in the membrane of all the cells, and therefore allows better visualization of cellular morphology. Lgr5-eGFP-IRES-CreERT2 mice were crossed with B6 Cg-Gt (ROSA)26Sortm9(CAG-tdTomato) Hze/J mice (Jackson Laboratories) to generate the Lgr5-eGFP-IRES-CreERT2; the Rosa26-CAG-tdTomato heterozygote was used for lineage tracing experiments. All the mice were maintained ad libitum, and all the studies were performed under the guidelines and protocols of the Institutional Animal Care and Use Committee of the University of Kansas Medical Center. All the animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center (ACUP number 2019-2487).

Lgr5-eGFP-IRES-CreERT2 mice were crossed with B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mice (Jackson Laboratories) [22 (link)] to generate the Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato heterozygote. To examine the contribution of Lgr5 ISC to tissue regeneration under steady-state conditions, lineage tracing was induced by tamoxifen administration in Cre reporter mice to mark the ISC and their respective tdT-positive progeny. Adult mice were injected with tamoxifen (Sigma; 9 mg per 40 g body weight, intraperitoneally) to label Lgr5⁺ lineages. For irradiation injury studies, mice were given 14–15 Gy AIR, and tissue was harvested on day 8 post-irradiation.