Fig. 4

Fmn exon-specific gene-targeting approach for separately deleting exons 4 and 5. The positions of a neomycin resistance gene (neo) replacing the respective deleted Fmn exon as well as a herpes simplex virus thymidine kinase gene (TK) for positive/negative selection from the pPNT vector (23) are indicated. Arrows indicate the transcriptional directions of the neo and TK genes. Prior to electroporation, the targeting vectors were linearized at the indicated unique NotI (N) sites. B, BamHI; Cla, ClaI; P, PstI; RI, EcoRI; RV, EcoRV; X, XbaI. (A) Gene-targeting Fmn exon 4-deletion construct. The replacement-type targeting construct contains 3.5 kb of genomic DNA upstream of exon 4 and 4.8 kb of downstream homologous sequence. Homologous recombination within the Fmn locus would introduce the neo gene and delete 2.5 kb of genomic DNA containing Fmn exon 4. (B) Genetargeting Fmn exon 5-deletion construct. The replacement-type targeting construct contains 4.5 kb of genomic DNA upstream of exon 5 and 3 kb of downstream homologous sequence. Homologous recombination within the Fmn locus would introduce the neo gene and delete 1.1 kb of genomic DNA containing Fmn exon 5. (C) Southern blot analysis for targeted exon 4- and exon 5-deleted ES cell clones. DNA was isolated from targeted ES cell clones (lanes 1 and 3) and parental ES cells (lanes 2 and 4). As expected, the Xbal restriction fragment size change from 11 kb to 7 kb is seen in the targeted exon 4-deleted ES cell clone (lane 1) using the 0.3 kb 5′ outside probe (a) indicated in Fig. 4A. Similarly, the BamHI/ClaI restriction fragment size change from 9.5 kb to 4.5 kb is seen in the targeted exon 5-deleted ES cell clone (lane 3) using the 0.8 kb 3′ outside probe (b) indicated in Fig. 4B. (D) Homozygous Fmn exon 4- and exon 5-deletion mice lack DNA sequences for exons 4 and 5, respectively. DNA isolated from wild-type, heterozygous mutant, and homozygous mutant littermate animals were first genotyped by Southern blot analysis. Then the same DNA samples were hybridized with a probe specific for the deleted Fmn exon. Wild-type, heterozygous fmn^4KO, and homozygous fmn^4KO DNA samples (lanes 1, 2, and 3, respectively) digested with BamHI were hybridized with a Fmn exon 4 probe. As indicated in the wild-type allele diagram in Fig. 4B, a 9.5 kb BamHI-digested DNA fragment is detected using a Fmn exon 4 probe. Wild-type, heterozygous fmn^5KO, and homozygous fmn^5KO DNA samples (lanes 4, 5, and 6, respectively) digested with EcoRI were hybridized with a Fmn exon 5 probe. As shown in the wild-type allele diagrams in Fig. 4A and B, a Fmn exon 5 probe detects a 5.5 kb EcoRI-digested DNA fragment.