Fig. 1: Generation of Mcph1-dBR1 knock-in mice.

A The target strategy to disrupt the N-terminal BRCT domain by deleting exons 2 and 3 in ES cells. After homologous recombination, the targeted allele contains 5′ arm including exon 1 and 3′ arm containing sequence from exon 4 to exon 6. In between, a neomycin cassette (neo, purple box) flanked by two loxP sites (red triangle). One nucleobase adenine (A) (“V”) was inserted at the end of exon 1 (yellow coloured) to keep the truncated protein in frame. Two probes for the Southern blot analysis are indicated, 5P4 (blue box) and 3P4 (orange box). The former detects a fragment of 7.1 kb in the wildtype (WT) allele, 9.3 kb in the targeted (Tg) allele, and 8.2 kb in the knock-in (Ki) allele after genomic DNA digested with SacI. 3P4 detects a fragment of 11.6 kb in WT allele, 10.4 kb in Tg allele, and 9.2 kb in Ki allele after genomic DNA digested with EcoNI and AhdI. The black box represents targeting region; the white box represents exons. B Southern blot analysis of targeted ES cells and mice. The genomic DNA of WT, targeted ES clones 2–8E ( + /Tg), and + /Ki ES clones 2–8E-7G, which was obtained after pMC-Cre transfection, were digested by SacI, or EcoNI and AhdI, followed by hybridisation which probes 5P4 (upper panel) and 3P4 (lower panel), respectively. Southern blot analysis of the liver of + /Tg, +/Ki and Ki/Ki mice is also shown to confirm the mutant alleles in these mutant mice. C Upper panel: Mcph1 cDNA was amplified using primer Ex1-F located in exon 1 and Ex8-R located in exon 8 to produce an 838 bp fragment in WT cDNA and 625 bp fragment in Ki cDNA. Lower panel: Sequencing results of Mcph1^Ki/Ki cDNA containing insA before exon 4. D Protein lysates were extracted from WT and Mcph1^Ki/Ki brains at postnatal day 1 (P1). The MCPH1 protein was examined by Western blotting using an anti-MCPH1 antibody. β-actin was used as a loading control. (E) Position and sequence of the peptides representing the MCPH1 protein used for targeted MS analysis. Pep1 spans the area of BRCT1 domain. Pep2 and Pep3 span the middle domain. Lower panel shows MCPH1 peptide expression profile of the E13.5 neocortex of control (n = 7), Mcph1-ΔBR1 (n = 4) and Mcph1-Δ mice (n = 4). Note the absence of MCPH1 Pep1, but remained Pep2 and Pep3 peptides in Mcph1-ΔBR1 neocortex and complete lacking MCPH1 peptides in Mcph1-Δ neocortex.