Alu RNA-transfected Primary Mouse Retinal Pigment Epithelium: A Pathologically Relevant In Vitro Model for...

Age-related macular degeneration (AMD), particularly non-exudative AMD, requires experimental models that better replicate human pathology. Current in vivo models remain technically demanding and time-intensive, whereas conventional in vitro systems fail to recapitulate disease-specific pathological triggers. Here, we present a method to establish a retinal pigment epithelial (RPE) degeneration model using primary mouse RPE cells transfected with Alu RNA, a retrotransposon directly implicated in geographic atrophy pathology. This protocol details the enzymatic isolation of primary mouse RPE cells, followed by Alu RNA transfection to induce RPE degeneration. Validation integrates morphological (hexagonal architecture), functional (polarity loss and mouse protein ZO-1 disruption), and molecular analysis (quantitative PCR). As a result, we observed multifactorial changes triggered by Alu RNA transfection: inflammatory cytokine secretion (mouse genes Ifn-β, Il-6, Tnf-α; p < 0.05) and cellular senescence (mouse genes p21 and p53 upregulation; p < 0.05). Compared to traditional acute stress models, this system recapitulates chronic inflammatory-degenerative cascades of AMD through standardized techniques, ensuring reproducibility. By combining aspects of simplified in vitro assays and complex in vivo models, this approach could serve as a preliminary platform for exploring retrotransposon-driven mechanisms and screening potential therapeutic candidates.