Semiquantitative Methods for GFP Immunohistochemistry and In Situ Hybridization to Evaluate AAV Transduction of Mouse Retinal Cells Following Subretinal Injection
| Autor: | Steven D. Sorden, Gennadiy Bondarenko, Brian J Christian, Alok K. Sharma, Sharron Webster |
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| Rok vydání: | 2020 |
| Předmět: |
Biodistribution
Genetic Vectors Green Fluorescent Proteins In situ hybridization Gene delivery Biology Toxicology Retina Pathology and Forensic Medicine Green fluorescent protein Mice 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine medicine Animals Tissue Distribution Molecular Biology In Situ Hybridization 030304 developmental biology 0303 health sciences Retinal pigment epithelium Retinal Cell Biology Dependovirus Immunohistochemistry Molecular biology Mice Inbred C57BL medicine.anatomical_structure chemistry 030220 oncology & carcinogenesis Female |
| Zdroj: | Toxicologic Pathology. 49:537-543 |
| ISSN: | 1533-1601 0192-6233 |
| Popis: | The goal of this study was to develop methods for the evaluation of green fluorescent protein (GFP) and GFP transcript biodistribution in paraformaldehyde-fixed paraffin-embedded (PFPE) eye sections to assess the effectiveness of Adeno-associated virus (AAV) gene delivery in an experimental ocular toxicity study. Female C57BL/6NTac mice were administered AAV2-enhancedGFP vector once via subretinal injection. One group also received anti-inflammatory therapy (meloxicam). Immunohistochemistry (IHC) and RNA in situ hybridization (ISH) for GFP were performed on PFPE serial eye sections and evaluated using semiquantitative methods. On day 43, GFP labeling in both IHC and ISH sections was greatest in the retinal pigment epithelium, compared with other retinal layers in which expression was negative to moderate. Despite the presence of IHC GFP labeling in the photoreceptor layer (PRL) in some animals, only low numbers of transduced cells were detected by ISH in the PRL. Simultaneous analysis of IHC and ISH may be needed for comprehensive assessment of gene transduction and protein biodistribution. This study demonstrates approaches for semiquantitative evaluation of IHC and ISH that allow interpretation and reporting of GFP expression in toxicity studies. |
| Databáze: | OpenAIRE |
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