Figure 3.

miR-33-Scaffolded shRNAs Promote Efficient Gene Silencing in Cultured Cells and Mice

(A) Relative reporter activity in HEK293 cells transfected with Fluc, PC-1, and SOD1 gene-silencing constructs and their sensor plasmids. (B) Firefly luciferase activities detected in treated mouse liver extracts. Mice were co-administrated with rAAV9-Fluc reporter vector at 1.0 × 10¹¹ GCs/mouse and the gene-silencing vectors rAAV9-shFluc (n = 5), shFluc^miR-33 (n = 5), or no shRNA (n = 4) at 5.0 × 10¹¹ GCs/mouse. After 3 weeks, mice were sacrificed to assess firefly luciferase activity. (C) pc-1 mRNA levels in the livers of mice treated with rAAV9-shPC-1 or rAAV9-shPC-1^miR-33. Five mice in the 1.0 × 10¹¹ GCs/mouse group and four mice in the 3.0 × 10¹⁰ GCs/mouse group. (D) apob mRNA levels in the livers of mice (n = 5 in each group) treated with rAAV9-shApob or rAAV9-shApob^miR-33. rAAVs at the indicated doses were injected into adult C57BL/6 mice by tail vein. After 3 weeks, mice were sacrificed for PC-1 and Apob gene expression analysis using qRT-PCR. shFluc is driven by the U6 promoter. shApob, shPC-1, and shSOD-1 are driven by the H1 promoter. Values are mean ± SD. GC, genome copy.