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| Caption | A three-lox recombination strategy was used to generate mutant mice presenting an allele deleted from exon 3 of the Epb4.1l3 gene, Epb41l3tm1.1Jglt (protein 4.1BKO3). A. Generation of the Epb41l3tm1.1Jglt allele (protein 4.1BKO3). In a first step, A 17.0-kb Epb4.1l3 genomic clone including exons 2 and 3 was isolated from a mouse 129/Ola genomic library. A 9.5-kb NsiI-NotI fragment of this clone was used for the construction of the protein 4.1Blox3 targeting fragment. This fragment was subcloned in a modified pBR322 vector containing a NheI-SalI polylinker from pBluescript KS II (Stratagene). A 1.0-kb ApaI-ApaI fragment containing Epb4.1l3 (protein 4.1B) exon 3 was amplified by PCR using primers containing loxP sites (open triangles) and was inserted between 2 corresponding ApaI sites, flanking Epb4.1l3 (protein 4.1B) exon 3 of the 9.5-kb fragment. A 2.9-kb NheI-XbaI fragment containing a floxed PGKHygromycin/GFP cassette was inserted into a SpeI site 0.5-kb downstream of exon 3 in the same orientation of the Epb4.1l3 (protein 4.1B) gene. The NsiI-NotI protein 4.1Blox3 targeting fragment was electroporated into ES cell line E14 subclone IB10. Electroporated cells were plated on mouse embryonic fibroblasts and selected with hygromycin B. Hygromycin-resistant cells were trypsinized and GFP expressing cells were isolated and then plated on 96-well microplates by flow cytometric analysis. Homologous recombinants were identified by long-range PCR analysis. Four of six analyzed clones showed a correct karyotype. In the four diploid ES cell clones, 5' and 3' homologous recombination was confirmed by Southern blot analysis using digestions and probes. In a second step, germline chimeras (Protein 4.1Bflox3HygGFP/+) were generated by injection of Epb4.1l3 (protein 4.1B) mutant ES cells into C57BL/6 blastocysts and crossed with FVB/N mice to produce outbred heterozygous offspring. The genotypes of all offspring were analyzed by PCR or Southern blot analysis on tail-tip DNA. To generate Epb41l3tm1.1Jglt/Epb41l3+ (Protein 4.1BKO3/+) mice, protein 4.1Bflox3HygGFP/+ mice were crossed with EIIACre deletor mice. In the deriving double transgenic offspring tail DNA the Epb41l3tm1.1Jglt allele, and the Epb4.1l3 (protein 4.1B) allele with two loxP sites flanking the exon 3 of the gene (protein 4.1Bflox3 allele), were detected by PCR. Restriction sites used for screening ((S) SwaI, (Ns) NsiI, (B) BamHI), DNA fragments resulting from digestions (double-headed arrows) and hybridization probes (A, B and Hygro (black boxes)) are indicated. Also depicted are the PCR primers DAL1afor (5'- TTCATTTGGTGAGATCATGG-3'), DAL1arev (5'-CTACCCGAAGAAGTGACTGG-3') and DAL1bfor (5'-TAAAGTACGCTTTGCTCTAC-3') that detected the different alleles. | ||||
| Copyright | This image is from Cifuentes-Diaz C, PLoS One 2011;6(9):e25043, and is displayed under the terms of the Creative Commons Attribution 4.0 International License. J:177866 | ||||
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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 04/16/2024 MGI 6.23 |
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