Journal: Journal of Thrombosis and Haemostasis

Article Title: An engineered U1 small nuclear RNA rescues splicing-defective coagulation F7 gene expression in mice

doi: 10.1111/jth.12471

Figure Lengend Snippet: U1+5a-mediated rescue of hFVII expression by AAV delivery. (A) Plasma hFVII antigen levels (mean and 95% confidence intervals) in mice (four mice per group) injected with the AAV2-FVII+5A and, 2 weeks later (arrow), with the AAV8-U1+5a viral vectors at the doses reported in the table. *Denotes the last time-point for this group of mice ( n = 7). Inset: scheme of the sequential injection protocol. (B) Analysis of hFVII mRNA forms in mouse liver. The aberrant (a) and normal (n) transcripts are depicted on the right. Arrows indicate primers used for RT-PCR. Electrophoretic separation of amplicons and analysis was as in Fig. . Mice were injected with 1.2 × 10 12 vector genomes (vg) per mouse of AAV2-FVII+5A and 6 × 10 11 or 1.2 × 10 11 vg per mouse of AAV8-U1+5a. M, size marker; bp, base pairs. (C) Representative examples of liver sections from mice treated as indicated. Cells were stained with a species-specific anti-hFVII antibody (green) and cell nuclei (blue) with DAPI. Stained sections were captured as indicated in Fig. . Scale bar, 50 μm.

Article Snippet: Sections were rinsed three times with PBS solution (10 min each) and incubated overnight at 4 °C with a polyclonal sheep anti-human FVII FITC conjugated antibody (2 μg mL −1 in 1%FBS/PBS) (Affinity Biologicals, Ancaster, ON, Canada).

Techniques: Expressing, Clinical Proteomics, Injection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Marker, Staining

Journal: Journal of Thrombosis and Haemostasis

Article Title: An engineered U1 small nuclear RNA rescues splicing-defective coagulation F7 gene expression in mice

doi: 10.1111/jth.12471

Figure Lengend Snippet: Kaplan–Meier survival curves (A) and ALT levels (B) in relationship with the AAV8-U1+5a dose injected. Mice ( n = 2–4 per group) were injected with AAV vector by the intravascular route at day 0. The vector doses per mouse are indicated. The open circle in panel B indicates mice injected with 6 × 10 12 vg per mouse of AAV2-FVII+5A and 1.2 × 10 11 vg per mouse of AAV8-U1+5a. The P values refer to statistical differences from a given ALT value and the baseline values. ALT, alanine aminotransferase enzyme.

Article Snippet: Sections were rinsed three times with PBS solution (10 min each) and incubated overnight at 4 °C with a polyclonal sheep anti-human FVII FITC conjugated antibody (2 μg mL −1 in 1%FBS/PBS) (Affinity Biologicals, Ancaster, ON, Canada).

Techniques: Injection, Plasmid Preparation