Figure 4

Recombination status of floxed Brca1 alleles in mutant osteoclasts from long bones and cell cultures. (a) Diagram indicating the position and orientation (arrowheads) of PCR primers relative to LoxP sites flanking Brca1 exon 11 as previously published.¹ Primer pair a/b was used to amplify a 530-bp genomic DNA fragment from floxed Brca1 alleles that did not undergo Cre-mediated recombination, whereas primer pair e/d was used to amplify a 647-bp fragment from such alleles after undergoing Cre-mediated recombination. (b) DNA was extracted from duplicate samples (obtained from different mice in each case) from either 2-mm sections of wild-type and mutant femoral bone or cultured osteoclasts, and amplified with either primer pairs a/b or e/d. DNA obtained from a mutant ovary known to harbor recombinant Brca1 alleles was used as control. The PCR products were electrophoresed on agarose gels and visualized under UV after staining with ethidium bromide. Although the expected 530 bp unrearranged Brca1 allele (primer pair a/b) was detected in all tissues examined, the 647-bp amplification product expected from the rearranged allele (primer pair e/d) was not seen in DNA extracted from either osteoclast-enriched secondary spongiosa or in vitro culture.