Abstract
Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of considerable interest. Such studies often seek to express tagged proteins to characterize their activities, localizations, and protein-protein interactions. Here, we describe a robust method for transient protein expression in macrophages using mRNA lipoplex transfections.
Keywords: mRNA transfection, Macrophage, Protein expression
Background
Typical methods to achieve protein expression, including transfecting DNA using cationic polymers, nucleofection, and viral transduction (Zhang et al., 2009), are particularly difficult in macrophages, as these cells have a potent immune response to various danger signals such as cytosolic DNA. Therefore, conventional methods for exogenous gene delivery result in poor transfection efficiency and cell death. We reasoned that transfecting mRNA, instead of DNA, would be a better alternative to achieve protein expression in macrophages, as suggested by recent reports (Van De Parre et al., 2004; McLenachan et al., 2013). We were able to achieve high transfection efficiencies without loss of macrophage viability (Koster et al., 2017). Our method does not require expensive equipment and can be adapted for expressing exogenous and endogenous proteins.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
To quantify the EGFP signal in the fluorescent cells (Step B11, Figure 3), a basic workflow is described below using NIS-Elements version 4.40. Figure 3. Expression of EGFP in RAW264.7 macrophages. Cells were transfected with 200 ng of EGFP mRNA. A. The EGFP fluorescent cells were visualized using a fluorescent microscope. B. A bright-field image of the cells. Scale bars = 20 μm.
Recipes
Acknowledgments
This work was funded by the NIH/NIAID (R01 AI087682, 1R01 AI130454, and R21 AI128427), Potts Memorial Foundation, and Washington University in St Louis School of Medicine. The authors have no conflicts of interest. This protocol was used for studies reported in Koster et al. (2017).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.