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Updated: Jan 3, 2026
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Published on: November 9, 2019
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Marc Herb1, Alina Farid1, Alexander Gluschko1
1Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne.
Abstract:
Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.
Insights
This study presents an efficient method for transfecting macrophages, overcoming challenges posed by pattern recognition receptors (PRRs). The protocol enables high mRNA transfection rates in primary macrophages without toxicity.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Macrophages utilize pattern recognition receptors (PRRs) to detect non-self molecules.
- PRR recognition complicates nucleic acid delivery, leading to activation, degradation, or cell death.
- Efficient transfection of primary macrophages remains a significant challenge in research.
Purpose of the Study:
- To develop a protocol for highly efficient mRNA transfection in murine primary macrophages.
- To overcome PRR-mediated immune responses that hinder transfection.
- To provide a reliable method for delivering mRNA into peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM).
Main Methods:
- In vitro transcription of mRNA from DNA templates (e.g., plasmids).
- Detailed description of mRNA generation and transfection procedures.
- Utilized primary murine macrophages, including peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM).
Main Results:
- Achieved high transfection rates: approximately 50-65% for PM and 85% for BMDM.
- Demonstrated absence of cytotoxicity and immunogenicity associated with the transfection protocol.
- Successfully delivered in vitro transcribed mRNA into primary macrophages.
Conclusions:
- The developed protocol enables efficient mRNA transfection in primary murine macrophages.
- This method circumvents PRR-mediated activation and degradation, improving experimental outcomes.
- The protocol offers a valuable tool for macrophage research, facilitating gene expression studies.
