Figure 4
In vivo deletion of the Acan upstream enhancer. (a) Genomic structure of the mouse Acan gene, consisting of 17 exons (black boxes). Black arrowheads indicate the positions of the upstream enhancer (UE), located approximately 10 kb upstream of the transcription start site, and of the I12 enhancer (I12E) located in intron 12. (b) The mouse DNA sequence surrounding the UE. The sequence of the UE is shown in bold letters. Two potential binding sites for PAX2/5/8, predicted by MatInspector software (Genomatix), are underlined. The shaded letters indicate the region of a deletion mutation produced by the CRISPR/Cas system. The boxed letters indicate the sgRNA target sequence. The bases comprising the SOX9-binding sites are indicated with asterisks. (c,d) Total RNA was extracted from the AF (c) and the costal cartilage (d) of UE+/+, UEd/+, or UEd/d male mice at 3 weeks after birth. Relative expression levels of Acan and Sox9 were examined by qRT-PCR analysis. qRT-PCR data represent the average of 4 mice in each group. The relative expression of each gene is normalized to that of UE+/+ and reported as mean ± s.d. *P < 0.05 versus UE+/+, **P < 0.01 versus UE+/+, ***P < 0.001 versus UE+/+.
