A Fibroblast-Based Adenoviral Reporter System Driven by the Mouse Collagen Type I Alpha 1 Promoter for Antifibrotic Drug Screening

Summary

This protocol describes the development of a fibroblast-based adenoviral reporter system driven by a mouse type I collagen promoter to enable longitudinal monitoring of fibroblast activation and facilitate high-throughput screening of antifibrotic compounds.

Abstract

Cardiac fibrosis, characterized by aberrant fibroblast activation and excessive extracellular matrix deposition, lacks target-specific therapies, largely due to the absence of longitudinal, scalable, and non-destructive in vitro screening platforms. Traditional end-point assays and resource-intensive stem cell models inherently preclude real-time monitoring of fibrotic progression. To overcome these limitations, this protocol describes the generation, optimization, and validation of a mouse collagen type I alpha 1 (Col1a1) promoter-driven adenoviral mCherry fluorescent reporter system (Ad-mCol1a1p-mCherry) in NIH/3T3 fibroblasts. The critical steps for recombinant adenovirus packaging, transduction optimization (multiplicity of infection) to minimize cytotoxicity, and the establishment of a robust transforming growth factor beta (TGF-β)-induced fibrosis model are detailed. By circumventing the need for cell fixation, this system enables direct and longitudinal monitoring of collagen transcription in live cells. The model’s specificity and reliability are pharmacologically validated using the TGF‑β type I receptor (ALK5) inhibitor SB431542, with fluorescent readouts correlating with endogenous fibrotic markers quantified via reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Ultimately, this cost-effective platform provides an accessible tool for the high-throughput screening of novel antifibrotic agents, thereby accelerating translational cardiovascular research.

Introduction

The management of cardiac fibrosis presents significant clinical challenges. Characterized by the aberrant activation of quiescent fibroblasts into myofibroblasts and excessive extracellular matrix (ECM) deposition, it represents an irreversible terminal pathway shared by heart failure, myocardial infarction, and other cardiovascular diseases^1,2,3. However, current treatment strategies face significant limitations. Conventional interventions—such as diuretics, β-blockers, and renin–angiotensin–aldosterone system inhibitors—primarily alleviate syst....

Protocol

We confirm that all experiments were performed in accordance with institutional and national guidelines. Because this study exclusively utilized the established NIH Swiss mouse embryo fibroblast cell line (NIH/3T3) and the mCol1a1p-mCherry reporter system, and did not involve any vertebrate animals, embryos, or human subjects, ethical approval from an institutional review board was not required for the procedures described in this manuscript.

All procedures involving recombinant adenoviral vectors were conducted in accordance with institutional biosafety regulations and standard Biosafety Level 2 (BSL-2) practices. Viral h....

Results

A schematic overview of the construction and packaging of the mCol1a1p-mCherry recombinant adenovirus is shown in Figure 1A. The mouse Col1a1 promoter was cloned upstream of the mCherry reporter gene and incorporated into an adenoviral vector, followed by viral packaging and amplification in HEK293 cells. The workflow illustrates key experimental steps, including viral infection, fibrotic stimulation, pharmacological intervention, and fluorescence-based si.......

Discussion

Cardiac fibrosis contributes to the progression of heart failure, and the development of effective antifibrotic therapies is limited by the lack of longitudinal screening platforms^2,13. Conventional methods for evaluating antifibrotic efficacy, including Western blotting, RT-qPCR, and immunofluorescence staining, rely on endpoint measurements that require cell lysis or fixation. These approaches prevent real-time monitoring of fibrotic progression in living cells.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
384-Well Glass-Bottom Microplate	Cellvis, USA	P384-1.5H-N	384-well glass-bottom plate used for cell seeding, viral infection, staining, and fluorescence imaging.
Adenoviral Backbone Plasmid	Microbix, Canada	pBHGloxΔE1,3Cre	Plasmid used to generate the full-length recombinant adenoviral genome.
Adenoviral Shuttle Vector: pDC315-mCol1a1p-mCherry	GeneChem, China	Custom service	Recombinant adenoviral shuttle vector containing the 2354 bp mouse Col1a1 promoter driving mCherry expression.
Benzonase Nuclease	Merck Millipore, USA	70664-3	Endonuclease used during adenoviral purification to remove contaminating nucleic acids.
Biological Safety Cabinet (Class II)	Esco Lifesciences Group, Singapore	AC2-4S1	Certified biosafety cabinet used for recombinant adenoviral handling and sterile cell culture procedures.
Cell Culture Medium (Complete DMEM/F12)	HyClone, USA	12800-017	Complete culture medium used for NIH/3T3 and HEK293 cell maintenance.
CellPathfinder Image Analysis Software	Yokogawa Electric Corporation, Japan	Version 3.08.01	Image-analysis software used for automated fluorescence quantification and cell segmentation.
CellVoyager CV8000 High-Content Screening System	Yokogawa Electric Corporation, Japan	CV8000	High-content imaging system used for mCherry and Hoechst fluorescence acquisition.
CO2 Cell Culture Incubator	Thermo Fisher Scientific, USA	3111	Humidified incubator maintained at 37 °C with 5% CO2 for mammalian cell culture.
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich, USA	D2650	Solvent used for SB431542 preparation.
Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)	Servicebio, China	G4610	Basal culture medium used for NIH/3T3 fibroblasts.
Fetal Bovine Serum (FBS)	Ausbian, Australia	VS500T	Serum supplement used at 10% (v/v) for complete culture medium preparation.
Hoechst 33342 Fluorescent Stain	Invitrogen, USA	H3570	Nuclear stain used for fluorescence imaging and nuclear count normalization.
HEK293 Cells	ATCC, USA	CRL-1573	Packaging cell line used for adenoviral production and amplification.
Human Recombinant TGF-β1	MedChemExpress, China	HY-P78214	Cytokine used to induce fibrotic activation in NIH/3T3 fibroblasts.
Image Acquisition Software	Yokogawa Electric Corporation, Japan	Version 3.08.01.03	Software used for automated fluorescence image acquisition and microscope control.
Lipid-Based Transfection Reagent	Invitrogen, USA	11668-019	Transfection reagent used for adenoviral plasmid delivery into HEK293 cells.
Microcentrifuge Tubes	Axygen, USA	MCT-150-C	Tubes used for RNA lysate storage and sample processing.
Microplate Reader	Tecan, Switzerland	Spark	Multimode microplate reader used for ELISA absorbance measurements.
Mouse Type I Collagen ELISA Kit	Meike Biotechnology, China	MK6778A	ELISA kit used to quantify secreted type I collagen in culture supernatants.
NIH/3T3 Mouse Fibroblasts	Procell Life Science, China	CL-0171	Mouse fibroblast cell line used for reporter model establishment and validation.
PacI Restriction Enzyme	New England Biolabs, USA	R0547S	Restriction enzyme used for adenoviral plasmid linearization.
Penicillin–Streptomycin Solution	Gibco, USA	15140-122	Antibiotic solution supplemented at 1% (v/v) to prevent bacterial contamination.
PerfectStart Green qPCR SuperMix	TransGen Biotech, China	AQ602	SYBR Green-based qPCR master mix used for quantitative PCR analysis.
Phosphate-Buffered Saline (PBS)	Servicebio, China	G4202	Buffer used for washing cells and reagent preparation.
pDC315-EGFP Shuttle Vector	GeneChem, China	N/A	Parent adenoviral shuttle vector used for reporter plasmid construction.
qPCR System	Applied Biosystems, USA	QuantStudio 5	Real-time PCR system used for quantitative gene expression analysis.
Restriction Enzymes (BamHI and KpnI)	Abclonal, China	RK21101, RK21104	Restriction enzymes used for shuttle-vector linearization.
Reverse Transcription Kit	TransGen Biotech, China	AT311	Reverse-transcription kit used for cDNA synthesis before RT-qPCR.
RNA Lysis Reagent	Invitrogen, USA	15596026	Reagent used for total RNA extraction from cultured cells.
SB431542	MedChemExpress, China	301836-41-9	TGF-β type I receptor inhibitor used for pharmacological inhibition experiments.
Sanger Sequencing Service/Reagents	Shanghai Boshang Biotechnology Co., Ltd., China	Custom Service	Sequencing service/reagents used for plasmid sequence verification.
Serological Pipettes	Corning, USA	4488	Sterile pipettes used for liquid handling during cell culture procedures.
T30 Thermal Cycler	LongGene Scientific Instruments, China	T30	Thermal cycler used for reverse transcription and PCR amplification.
Trypsin-EDTA Solution	Gibco, USA	25200-056	Cell dissociation reagent used for routine cell passaging.
Viral Purification Kit	Takara Bio USA	631533	Kit used for recombinant adenovirus purification.
Water Bath	Shanghai Yiheng Scientific Instruments,China	HWS-12	Temperature-controlled water bath used during adenoviral freeze–thaw cycles.