Alu RNA-transfected Primary Mouse Retinal Pigment Epithelium: A Pathologically Relevant In Vitro Model for Age-related Macular Degeneration

Summary

In this protocol, we describe the full process of establishing a pathology-relevant model for age-related macular degeneration (AMD) in primary mouse retinal pigment epithelial (RPE) cells using Alu RNA transfection.

Abstract

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.

Introduction

Age-related macular degeneration (AMD) is a predominant cause of global visual impairment. However, effective treatments for non-exudative AMD remain limited. Thus, there is an urgent need for reproducible and efficient research models to uncover disease mechanisms and identify therapeutic targets^1,2. Central to this effort is the retinal pigment epithelium (RPE), a monolayer situated between Bruch's membrane (choroid) and photoreceptors (retina), which plays a pivotal role in sustaining retinal homeostasis by supporting photoreceptor function, regulating the blood-retinal barrier, and facilitating waste c....

Protocol

This study was approved by the Animal Ethics Committee of Shanghai General Hospital affiliated to Shanghai Jiao Tong University School of Medicine. This protocol strictly follows the Regulations for the Administration of Affairs Concerning Experimental Animals (issued by the State Science and Technology Commission) and the guidelines for the ethical use of animals in ophthalmic and vision research established by the Association for Research in Vision and Ophthalmology (ARVO). See the Table of Materials for more details related to the materials used in this protocol.

1. Isolation of primary mouse RPE cells

....

Results

Primary mouse RPE cells before Alu RNA transfection
On Day 0 (Figure 1), primary mouse RPE cells were isolated and exhibited the characteristic hexagonal morphology, which is typical of healthy RPE cells. These cells maintained a well-organized monolayer. The cells were cultured to allow for initial structural organization and formation of tight junctions, as evidenced by the hexagonal shape and well-defined boundaries. This morphology served as the baseline for the subs.......

Discussion

Our goal was to establish a reproducible and accessible RPE injury model that enables high-resolution imaging and mechanistic analysis with minimal resource constraints. This approach provided clear visualization of Alu RNA-induced structural disruption comparing with control group, including apical tight junction fragmentation and basal cytoskeletal disorganization through confocal Z-stack imaging (0.5 µm step size). Two critical steps ensure the system's reliability: enzymatic digestion and Alu RNA transfectio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1300 Series Class II, Type A2 Biological Safety Cabinet	Thermo Scientific
37 °C/5% CO2 incubator	Thermo Scientific
4% Paraformaldehyde (PFA)	Servicebio	G1101-500ML
human Alu RNA			Gifted by L.Z.
Anti-rabbit secondary antibody,Alexa Fluor 568(goat)	Invitrogen Thermo Scientific	A11011
Applied Biosystems MicroAmp adhesive optical cover	Thermo Fisher Scientific
Applied Biosystems MicroAmp Optical 96-Well Reaction Plates	Thermo Fisher Scientific
Applied Biosystems ViiA 7 Real-Time PCR System	Thermo Fisher Scientific		Real-time PCR system for quantitative gene expression analysis
Bovine Serum Albumin(BSA, Fraction V)	Beyotine	ST023-50g
Carl Zeiss Primovert inverted cell culture microscope	Zeiss		For cell morphology monitoring
Centrifuge 5424 R	eppendorf
Chloroform	Sigma		Phase separation in RNA extraction.
Confocal laser scanning microscope(LSM900+Cell discover 7)	Zeiss		High-resolution imaging of immunofluorescence markers (e.g., ZO-1 and F-actin).
Costar 24/48/96 well plate	Corning
coverslip	Thermo Fisher Scientific		Used on 24-well plate for immunofluorescence imaging.
DAPI	Thermo	62248
Digital dry bath	Mulab
DMEM/F12	gibco		Media to grow RPE cells
Dulbecco's Phosphate-Buffered(D-PBS)	ShareBio	SB-CR017
Earle’s Balanced Salt Solution (EBSS)	gibco		Balanced salt solution used for papain and DNase I dilution during RPE isolation.
Ethanol (Absolute)	Sigma		RNA precipitation and column washing.
Fetal Bovine Serum (FBS)	Sigma		For complete RPE cell culture media
forceps			For dissection
Lipofectamine 3000 (Cationic Lipid-Based Transfection Reagent)	Thermo Fisher	L300015	To transfect Alu RNA into RPE cells
matrigel	Corning	356234	Matrix (1.14% dilution) for coating plates to support RPE adhesion and polarization.
Methanol-Free Formaldehyde	Thermo Scientific		Alternative fixative (4%) for preserving epitopes in immunofluorescence
Micropipette	eppendorf
Nanodrop 2000 spectrophotometer	Thermo Scientific		Measures RNA concentration and purity
Nikon ECLIPSE Ti-S fluorescent Inverted microscope	Nikon		Fluorescence imaging of stained samples
Olympus SZX16 stereomicroscope	Olympus		For dissection of mouse eyeballs and RPE isolation
Opti-MEM	gibco		low-serum medium for transfection complex preparation
Papain dissociation system	Worthington	LK003150	For working enzyme
Penicillin-Streptomycin	gibco		For complete RPE cell culture media
Pentobarbital sodium salt	Sigma		Euthanasia agent
Phalloidin 488	Abcam	176753	For polarity assessing
PrimeScript RT reagent Kit	TaKaRa	RR037A	Reverse transcription kit for cDNA synthesis (qPCR template preparation)
RNAsimple Total RNA Kit	TIANGEN	DP419	For extracting RNA
TB Green Premix Ex Taq (Tli RNaseH Plus)	TaKaRa	RR420A	For qPCR
ZO-1 Rabbit Polyclonal Antibody	Proteintech	21773	For morphological evaluation