A Novel Bone Morphogenetic Protein 2 Mutant Mouse, , Displays Impaired Intracellular Handling in Skeletal Muscle
Figure 2
Integration of thetargeting vector. (a) Schematic of the targeting vector recombining into genomic DNA. The BMP2 exons are numbered and represented as black boxes, while the neo cassette and thymidine kinase (TK) gene are represented as grey boxes. Insertion of the neo cassette introduces a DrdI restriction site as indicated. The mutation of the bipartite NLS (KREKRQAKHKQRKRLKS to KREKRQAKHKQAAALKS) in exon 3 is denoted by AAA. (b) After a DrdI digest and Southern hybridization using a 3′ flanking probe complementary to the region indicated with the striped box, the wild type band was 13.8 kb and the targeted allele band was 6.1 kb. (c) To verify male germline self-excision of the neo cassette, PCR was performed using primers that bound to BMP2 outside the neo cassette. PCR produced a 525 bp band from wild type DNA and a 607 bp if the targeting vector was inserted and the neo cassette excised, leaving a LoxP sequence. (d) Expression levels of conventional BMP2 were measured in skeletal muscle by western blot of cytoplasmic extracts using an anti-BMP2 antibody. The bands representing BMP2 proprotein, precursor of secreted BMP2, were quantified digitally and mutants were normalized to wild types within each experiment before averaging between experiments.for wild type gastrocnemius,for mutant gastrocnemius, andfor both wild type and mutant quadriceps.
