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. 2020 Aug 18;48(21):11827–11844. doi: 10.1093/nar/gkaa670

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 10. — Exogenously expressed Ago2 loads F12 siRNA (siF12-1, 3 mg/kg) 1–3 weeks after dosing. An AAV vector was designed to express 3xFLAG-tagged mouse Ago2 (FLAG-mAgo2) under a constitutive liver-specific promoter (TBG). (A) Expression of the FLAG-mAgo2 protein was verified in AAV treated mice by Western Blot using an anti-FLAG antibody. Days refer to days post siRNA dose (day 0). FLAG-mAgo2 AAV was injected into three cohorts of mice on day 7 of the experiment (B) Plasma F12 protein levels were quantified by ELISA and normalized to pre-dose for each animal. (C) F12 antisense siRNA liver levels were quantified by RT-qPCR. (D) RISC loading was performed by anti-FLAG IP for both -AAV and +AAV groups (FLAG-mAgo2) and by Ago-APP for -AAV and +AAV groups (Total Ago (APP)). Loaded antisense siRNA was quantified by RT-qPCR. Data is represented as mean ± SD, for N = 3. Welch's independent t-test was utilized to compare the mean value between the control and +AAV groups at each timepoint in (C, D), with statistically significant increases in either liver level or RISC loading shown for the +AAV groups (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Full results for the statistical analysis are included in the Supplemental Information.