Generation and Characterization of Transgenic Mouse Models

Generation of Kit-KO, Kit-EGFP, and Sox2^loxP/loxP mice have been previously described [8 (link), 37 (link), 41 (link)]. Sox2^loxP/loxP were intercrossed with Rosa26Cre-Ert2 mice (Jackson Laboratories, Ann Harbor, MI) to generate Sox2^loxP/loxP, Rosa26Cre-Ert2 mice. Sox2^loxP/loxP Rosa26Cre-ERT mice were crossed to p18 mice to generate Sox2^loxP/loxP, Rosa26Cre-Ert2 Kit-EGFP (p18) mice. Kit-KO mice were intercrossed with p18 to generate Kit-KO, Kit-EGFP. For embryo staging, 0.5 dpc corresponded to the day of vaginal plug. Knockin mice carrying the KitD814Y mutation (see below) were generated by injecting mutant ESCs into C57/B6 carrier blastocysts and transplanted the same day into foster CD1 mothers. All experiments were performed in compliance with the Tor Vergata University Institutional Animal Care and National Institutes of Health Intramural Animal Care and Use program. All procedures adhered to the standards published in Guide for the Care and Use of Laboratory Animals. Experimental procedures involving mice were approved by the Italian Ministry of Health. The structure of the targeting construct used to generate KitD814Y knockin ESCs is depicted below.