Notch dimerization and gene dosage are important for normal heart development, intestinal stem cell maintenance, and splenic marginal zone B-cell homeostasis during mite infestation
Fig 1
Generation of Notch dimerization-deficient mice.
A. Electrophoresis mobility shift assay for purified proteins binding to CSL (magenta) or SPS (green) probes. Balls mark occupancy of 1 or 2 sites. Note that cooperative binding by WT NTC, but not the RA mutant NTC, specifically depletes the SPS probe but not the CSL probe (compare lanes 8 and 13). See text and S1 Fig for detail. B-C. CRISPR-Cas9-mediated double-strand break was used to mediate homologous recombination of a short oligonucleotide into Exon 32 substituting Arg N1R1974 (B-B”) and N2R1934 into Ala (C-C”), generating N1RA/RA and N2RA/RA animals. To facilitate genotyping, 2 silent mutations (in red) were included in the oligo, abolishing the BglII restriction site while generating an XbaI site in Notch1 (B’). In Notch2, a silent mutation (in green) was included to create a BglII site (C’). Sequencing PCR products containing these regions confirmed the presence of the Arg to Ala substitution in founders; digestion of these PCR products with XbaI (for N1, B”) and BglII (for N2, C”) confirmed the presence of 1 (N1) or 2 (N2) mutant alleles. See S1 Data for raw data. CSL, CBF1/Suppressor of Hairless/LAG-1; MAML, mastermind-like; N1ICD, Notch1 intracellular domain; N1RA/RA; N2RA/RA, Notch1/Notch2 RA homozygous; NTC, Notch transcription complex; RA, Arg (N1R1974/N2R1934) to Ala substitution; RBPj, recombinant binding protein for immunoglobulin Kappa j region; SPS, sequence-paired site; WT, wild-type.