Opposite Phenotypes of Muscle Strength and Locomotor Function in Mouse Models of Partial Trisomy and Monosomy 21 for the Proximal Hspa13-App Region (original) (raw)
Fig 1
Generating a 9.2 Mb deletion and reciprocal tandem duplication between the Hspa13 and App loci.
(A) The 9.2 Mb targeted region defined by the Hspa13 and App genes as shown from a capture derived from the UCSC genome browser (http://genome.ucsc.edu/). Black headband: chromosomes bands; blue headband: contigs; red rectangle boxes: protein coding genes. (B) The targeting vectors containing a loxP site (green arrow), a selectable antibiotic resistance gene (puro or neo), and a part of the Hprt gene (3’ or 5’ Hprt, red arrows) were integrated successively in the Hspa13 locus (Hspa13tm1Yah) and in the App gene (Apptm1Yah). The start and end positions of the genomic homologous regions present in the 5’ and 3’-Hprt vectors are from mouse genome assembly GRm38. The Stchtm1Yah allele was checked by Southern blot analysis. The digestion of DNA with SacI (S) restriction enzyme and hybridisation with external probes A and B gives additional fragments of 19.7 kb and 14.3 kb, respectively, for the Stchtm1Yah allele compared to the wild-type (wt) allele of 17.2 kb. The hybridisation of the same DNA digest with the Gap1 probe gives two additional fragments of 19.7 and 14.3 kb for the Hspa13tm1Yah. (C) The integration of the 3’Hprt vector was confirmed in the same manner by digestion with the _Psi_I (P) enzyme and hybridisation with probes C (wt, 11.5 kb; Apptm1Yah allele, 10.9 kb), D (wt, 11.5 kb; Apptm1Yah allele, 9.9 kb), and Gap2 (wt, 11.5 kb; Apptm1Yah allele, 10.9 and 9.9 kb). (D) After Cre-mediated recombination, the deletion (Ms3Yah allele) and duplication (Ts3Yah allele) were identified by _Nde_I (N) digestion, and a probe was made on the ampicillin gene (Ampi) present in both targeting vectors that recognises recombinant clones with the Ms3Yah (23.2 kb) and Ts3Yah (18.2 kb) alleles from nonrecombinant ones that still carry the Hspa13tm1Yah (26 kb) and Apptm1Yah (15.4 kb) alleles. Puro: puromycin, neo: neomycin. (E) Interphase FISH analysis with BAC probes that map in the region of the deletion or duplication (BAC bMQ-34K13; red) and outside (BAC bMQ-381L17; green). The wild-type (diploid) showed two red and two green adjacent signals. On the other hand, nuclei from Ms3Yah showed two green and only one red signal due to the deletion of the Hspa13-App region, and nuclei from Ts3Yah showed two green and three red signals. (F) The CGH profile of Mmu16 was established using NimbleGen mouse HD2 whole genome CGH oligonucleotide arrays comprising 2,100,000 isothermal probes 50–75 bp length, with a median spacing of 1.1 kb throughout the genome (UCSC NCBI37/mm9/July 2007 assembly) enabling high-resolution CNV detection. It confirmed the loss of one copy of the Hspa13-App fragment in Ms3Yah mice and the presence of an additional copy of the same fragment in Ts3Yah animals. Plotted are log 2 transformed hybridisation ratios of Ms3Yah and Ts3Yah versus diploid mouse DNA.