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A three-lox recombination strategy was used to generate mutant mice presenting an allele deleted from exon 3 of the Epb4.1l3 gene, Epb41l3^tm1.1Jglt (protein 4.1B^KO3). A. Generation of the Epb41l3^tm1.1Jglt allele (protein 4.1B^KO3). 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 4.1B^lox3 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 4.1B^lox3 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 4.1B^{flox3HygGFP/+}) 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 Epb41l3^tm1.1Jglt/Epb41l3⁺ (Protein 4.1B^KO3/+) mice, protein 4.1B^flox3HygGFP/+ mice were crossed with EIIACre deletor mice. In the deriving double transgenic offspring tail DNA the Epb41l3^tm1.1Jglt allele, and the Epb4.1l3 (protein 4.1B) allele with two loxP sites flanking the exon 3 of the gene (protein 4.1B^flox3 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.

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

Associated
Alleles

Symbol	Name
Epb41l3^tm1.1Jglt	erythrocyte membrane protein band 4.1 like 3; targeted mutation 1.1, Jean-Antoine Girault

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