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. 2022 Mar 11;4:785698. doi: 10.3389/fgeed.2022.785698

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Copyright © 2022 Lisjak, De Caneva, Marais, Barbon, Biferi, Porro, Barzel, Zentilin, Kay, Mingozzi and Muro.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Gene targeting in adult wild-type mice liver with a reporter donor-eGFP. (A) Experimental scheme. WT mice were transduced with AAV8-SaCas9 (1.0E11 vg/mouse) and AAV8-donor-EGFP (5.0E11 vg/mouse) at postnatal day (P) 60 and their livers were collected 2 weeks post-injection (n = 3); (B) Western blot analysis of eGFP protein from liver extracts. In the analysis are included two samples from animals treated at P60 with donor-eGFP+Cas9, donor-eGFP (no Cas9), untreated wild-type mouse (−) and mouse treated with donor-eGFP+Cas9 at P2 (+); (C,D) rAAV8-SaCas9 and rAAV8-donor-eGFP viral genome copy analysis; (E) Immunohistochemical analysis of eGFP protein in mice treated with SaCas9 and donor-eGFP at P60 and P2. Nuclei were stained with Hoechst (blue). Scale bar, 200 μm; (F) Quantification of eGFP-positive cells normalized by the total number of cell nuclei.