Figure 1.

Generation of Lrp4 R1170W Knockin Mouse Model

(A) Mouse Lrp4 genomic DNA schematic, showing the exon 25/intron 25 targeting area. A CRISPR/Cas9 approach was used to replace a 135-bp sequence with a donor oligo containing a C→T mutation at position 1 of aa1170, as well as two silent mutations in the flanking codons to facilitate genotyping.

(B) PCR products from exon 25 were sequenced to confirm mutations. A representative Sanger electropherogram from a heterozygous knockin mouse shows equal expression of the three individual base substitutions in the three consecutive codons.

(C) Western blot of cortical bone tissue protein extract from 6- to 8-week-old Lrp4 WT and Lrp4^KI mice, immunoreacted for Lrp4 (∼240kDa), indicating comparable Lrp4 protein expression levels in Lrp4^KI versus Lrp4 WT mice. Lane loading equivalency was assessed by stripping and reprobing for the focal adhesion protein vinculin (lower panel) and via Ponceau-S staining of the membrane for total protein before blotting (right panel). Two control lanes were removed between molecular markers and wild-type lanes for clarity.