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. 2019 Jul 2;17(7):e3000350. doi: 10.1371/journal.pbio.3000350

Fig 3. Validation of Rln1 and Irx5 by generation of mutant mouse lines.

Fig 3

(A) Schemetic of Rln1 and Irx5 KO mouse line construction via zygote injection of Cas9 mRNA and sgRNA. (B) Whole-mount staining of WT and Rln1 KO mice (F1) at P0 by alcian blue and alizarin red staining. (C) μCT analysis of distal femoral metaphysis from 12-week-old WT and Rln1 KO mice. Representative three-dimensional reconstruction of μCT images (left) and the percentage of trabecular BV/TV (right). (D) Alcian blue and alizarin red staining of the whole skeletons, forelimb, hindlimb, calvarium, and clavicle of the newborn WT and Irx5 KO mice generated through zygote injection of CRISPR-Cas9. (E) μCT analysis of distal femurs from 4-week-old WT and Irx5 KO mice. Representative images of three-dimensional reconstruction of μCT. (F) BV/TV of Irx5 KO mice and control littermates. (G) Tb.N of Irx5 KO mice and control littermates. (H) Tb.Th of Irx5 KO mice and control littermates. (I) C.Th of Irx5 KO mice and control littermates. ***P < 0.001; **P < 0.01; *P < 0.05 versus WT. n.s., P > 0.05. Data associated with this figure can be found in S1 Data. BV/TV, bone volume per tissue volume; Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced palindromic repeats; C.Th, cortical thickness; KO, knockout; n.s., no significant difference; P0, postnatal day 0; sgRNA, single guide RNA; Tb.N, trabecular number; Tb.Th, trabecular thickness; WT, wild type; μCT, microcomputed tomography.