Fig 1: Rats with PLS3 E10-16del mutation built by CRISPR/Cas9 and confirmation.(A) Schematic diagram of targeted PLS3 gene deletion using CRISPR/Cas9 and Sanger sequencing analysis. Four specific CRISPR target sites near the genomic region of exon 10–16 were designed. CRISPR/Cas9 systems specifically cleaved the target sites. Sanger sequence confirmed the genomic deletion of exon 10–16 (No. 84172–93797 bp) and two base pair insertions at the target site due to non-homologous end joining after the DNA cleavage. (B) Polymerase chain reaction (PCR) genotyping of PLS3E10-16del/0 rats. DNA from tail snips was subjected to PCR using P-KO-F/P-KO-R and P-WT-F/P-WT-R primers for mutant and WT allele, respectively. Amplification of mutant samples resulted in one copy of the upper 554 bp fragment, while WT samples yield one copy of the lower 450 bp fragment. (C) PLS3 gene deletion confirmed by western blot. PLS3 protein expression was absent in various tissues of the PLS3E10-16del/0 rats compared to age-matched WT rats, indicating the whole-body deletion of the PLS3 gene. (D) Quantitative PCR (qPCR) confirmation of PLS3 E10-16 deletion. The results of PLS3-KO indicated the expression level of PLS3 E10-16 (knockout region). The results of PLS3 indicated the expression level of PLS3 E1-9 (uneditable region). The expression level of PLS3 E10-16 was extremely low in PLS3E10-16del/0 rats while the expression level of PLS3 exon 1–9 was similar to WT rats. Data were pooled from three independent experiments and were presented as mean ± SEM. Data were analyzed using unpaired two-tailed Student t test. *p <0.05 vs WT groups. (E) Representative image of PLS3 immunohistochemistry in the femoral sections. Os, osteocytes; Ob, osteoblasts; Oc, osteoclasts. Immunohistochemical staining images revealed that PLS3 was present in osteocytes, osteoblasts, and osteoclasts in the cortical (left) and trabecular (right) bone of WT rats. Loss of staining in osteocytes, osteoblasts, and osteoclasts in the PLS3E10-16del/0 rats confirmed the successful deletion of the PLS3 gene in bone. WT: wild-type. Figure 1—source data 1.Sanger sequencing confirmation of PLS3 E10-16 knockout. Figure 1—source data 2.Western blotting analysis of PLS3 protein expression. Figure 1—source data 3.Original data of quantitative polymerase chain reaction (qPCR) results.
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