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. 2023 Jan 19;55(2):232–245. doi: 10.1038/s41588-022-01280-z

Extended Data Fig. 3. Monoallelic and biallelic recombination at Notch1 locus results in reduction of Notch1 mRNA and protein.

Extended Data Fig. 3

a. Protocol for b, f-h. Highly induced YFPCreNotch1flox/flox or +/flox mice aged to allow Notch1 mutant cells to colonize epithelium. Controls, non-induced mice (+/+). b. Esophageal sections 10 days post induction (p.i.) stained for NOTCH1 (magenta), Wheat germ agglutinin (WGA) (gray) and DNA (blue). Scale bars, 30 µm. c. Quantitative PCR assay for Notch1 recombination. Primer set B measures floxed exon1, C amplifies recombined locus, A allows normalization. d, e. Validation using set B (d) and set C (e) against standard curve (Mean ± SEM, n = 3 technical replicates). f-h. YFPCreNotch1flox/flox or +/flox mice and controls aged for 8 weeks, mean ± SEM, each dot represents a mouse, n = 4 mice. f. Exon1/Exon3 ratio assay using set B. One-way ANOVA; adjusted p values from Tukey’s multiple comparisons test against wild type. g. Notch1:Gapdh mRNA by RT-qPCR. One-way ANOVA; adjusted p values from Tukey’s multiple comparisons test against wild type. h. Immune Capillary Electrophoresis of NOTCH1 transmembrane/intracellular domain (NTM1 + NICD1) and α-Tubulin protein. Dashed lines indicate image cropping and arrangement. One-way ANOVA; adjusted p values from Tukey’s multiple comparisons test against wild type. i. Protocol. YFPCreNotch1flox/flox or +/flox or +/+ mice were clonally induced, whole mounts stained for NOTCH1, YFP and DAPI. Clones were identified by NOTCH1 staining (Extended data Fig. 2a, Supplementary Note). j. Epithelium stained for NOTCH1 (magenta), YFP (green) and DNA (blue) 13 weeks p.i. of YFPCreNotch1flox/flox or +/flox or +/+ mice. Scale bars, 500 µm. k-n. Validation of clonal genotype. k. Protocol. Tissues were stained for NOTCH1, YFP and DNA at 4 weeks p.i. for YFPCreNotch1flox/flox and 13 weeks p.i for YFPCreNotch1+/flox. Potential clones were micro-dissected (yellow dotted lines) and qPCR performed using set C. l, m. Representative examples of 4 weeks post-induction (w.p.i.) Notch1−/− clones and control areas (upper panels) and 13 w.p.i. Notch1+/− clones and control areas (lower panels) validated as in k. NOTCH1, magenta, YFP, green, DNA, blue. l. Projected view. Dotted lines: orange, dissected clones, blue, control areas. Scale bars, 125 µm. m. (x, y) basal view. White dotted lines: clone edges. Scale bars, 30 µm. n. qPCR assay using primer set C (c, e). Mean± SEM, each dot represents a sample, n = 22 controls and n = 21+/− clones from 3 YFPCreNotch1+/flox mice; n = 21 controls and n = 22 −/− clones from 3 YFPCreNotch1flox/flox mice. Two-tailed unpaired Student t-test. AU, arbitrary units. SEM, standard error of mean. See Supplementary Table 6.

Source data.