Fig. 3. Targeted homologous disruption of the murine myoferlin (fer1L3) locus. (A) Gene structure of the first six exons of myoferlin, showing the start codon (ATG). Those regions that encode the C2A domain are shown in black. (B) A neomycin-containing cassette was used to replace the transcriptional and translational start site of myoferlin. The thick black bars represent the homology arms present in the targeting construct. (C) PCR confirmed the replacement of exon 1 with neomycin. (D) Immunofluorescence microscopy using the MYOF3 antibody reveals that myoferlin null myoblasts do not express myoferlin protein. Scale bar: 20 µm. (E) Partially differentiated cultures of primary myoblasts from myoferlin null mice expressed no myoferlin protein, as detected by immunoblotting with an anti-myoferlin antibody whose epitope does not reside in the deleted region. An increased level of dysferlin expression was noted, while levels of other membrane associated proteins, dystrophin and annexin II, were unchanged.