Scientific Reports. 2022 Dec 27
Yanxia Gao, Kailun Fang, Zixiang Yan, Haiwei Zhang, Guannan Geng, Weiwei Wu, Ding Xu, Heng Zhang, Na Zhong, Qifang Wang, Minqing Cai, Erwei Zuo, and Hui Yang
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AAV vector packaging Different serotypes of AAVs were packaged and titered by Gene Editing Core Facility in the Institute of Neuroscience or PackGene Company. Request Quote

Research Field: Eye

Keywords: Differentiation, Neurogenesis, Retina

AAV Serotype: AAV9

Dose: To test the labeling specification of different AAV serotypes, 1 μl of AAV including 1 × 10^9 or 1 × 10^8vg was separately injected. To test the labeling specifications of the hGFAP-Cre AAV vector, 1 μl of AAV including1 × 10^10, 1 × 10^9, or 1 × 10^8vg was separately injected. In gene transfer of transcription factors experiments, 1 μl of AAV included 5 × 10^8 vg hGFAP-Cre and 1 × 10^10 vg hGFAP-EGFP-WPRE in the control group, 5 × 10^8vg hGFAP-Cre and 1 × 10^10vg hGFAP-Math5-Brn3b-EGFP-WPRE, or 1 × 10^10vg hGFAP-NeuroD1-EGFP-WPRE, or U6-sgRNA(Ptbp1)-hGFAP-Cas13X in the overexpression or knockdown groups. All mice were injected at 4 ~ 8 weeks old.

Routes of Administration: AAVs were delivered to the eyes via subretinal injection

Targeted organ: eye

Animal or cell line strain: mice

Abstract

Reprogramming Müller glia (MG) into functional cells is considered a promising therapeutic strategy to treat ocular diseases and vision loss. However, current AAV-based system for MG-tracing was reported to have high leakage in recent studies. Here, we focused on reducing the leakage of AAV-based labeling systems and found that different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) removed. The leakage ratio of the AAV9-hGFAP-Cre-ΔWPRE decreased by an approximate 40-fold compared with the AAV9-hGFAP-Cre-WPRE labeling system. In addition, we validated the specificity of the AAV-ΔWPRE system for tracing MG reprogramming under Ptbp1-suppression and observed strict non-MG-conversion, similar to previous studies using genetic lineage tracking mouse models. Thus, the AAV9-hGFAP-Cre-ΔWPRE system showed high efficiency and specificity for MG labeling, providing a promising tool for tracing cell fate in vivo.

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