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| Caption | Generation of the Capn3tm1.1Hiso allele. (A) Targeting scheme for the p94KI construct. A missense mutation [6th T to A in exon 3 of the p94/calpain 3 gene, Capn3 (agGGGACTGCTGGTTT -> agGGGACAGCTGGTTT)], was introduced into exon 3 by performing PCR on a 1.7-kb BstPI fragment subcloned from 129S mouse genomic DNA. The missense mutation changed Cys129, an amino acid residue in the catalytic center of p94, to Ser, and created a PvuII site. The complete nucleotide sequence of the mutated 1.7-kb BstPI fragment was verified by DNA sequencing. A neomycin-resistance gene (neoR) flanked by loxP sequences was constructed as follows. A DNA fragment was generated by PCR using primers lox1s and lox1a and pMC1neo polyA+ vector DNA (Stratagene) as a template. The 3'-part of the fragment was rescued by cutting it with RsrII and BamHI, which resulted in a neoR-loxP 3' fragment of 255 bp, and the fragment was cloned into the 3.6-kb pMC1neo polyA+/RsrII-BamHI fragment (Plasmid A). The 5'- loxP part was amplified by performing half-nested PCR, first with primers lox2s-1 and lox2a and then with lox2s-2 and lox2a, on pMC1neo polyA+ plasmid DNA. The amplified PCR fragment was cloned into the TA TOPO cloning vector (Invitrogen), and the 5'-loxP-containing SpeI-ClaI fragment (SpeI-TOPO vector-NdeI-loxP-ClaI) was used for ligation with the XhoI-SpeI fragment (XhoI-neoR-loxP-NdeI-SpeI) of Plasmid A described above, resulting in loxP-neoR-loxP flanked by NdeI sites (NdeI-loxP-neoR-loxP-NdeI) in the TOPO vector. The neoR cassette was cut by NdeI and inserted into the NdeI site located 280-bp downstream of the exon 3-intron 3 junction of Capn3. The C129S mutation and neoR cassette insertion were then ligated together, and the HindIII-BglII fragment of pMCDT-A(A+T/pau) (Oriental Yeast) was added to the 3'-end of the final targeting vector. | ||||
| Copyright | This image is from Ojima K, J Clin Invest 2010 Aug 2;120(8):2672-83 and is displayed with the permission of the American Society for Clinical Investigation who owns the Copyright. J:163711 | ||||
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Associated Alleles |
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Mouse Genome Database (MGD), Gene Expression Database (GXD), Mouse Models of Human Cancer database (MMHCdb) (formerly Mouse Tumor Biology (MTB)), Gene Ontology (GO) |
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last database update 05/07/2024 MGI 6.23 |
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