This article describes a method for transfecting mouse-derived primary macrophages with modified mRNAs encoding green fluorescent proteins. The process enhances mRNA stability and prevents immune response, allowing for efficient protein synthesis within the cells.
Begin with a tube containing in vitro transcribed mRNAs that encode green fluorescent proteins.
The mRNA contains modified nucleotides, 5-methyl-cytidine triphosphate, and pseudouridine triphosphate, which prevent immune response and enhance mRNA stability.
The 5'-end of the mRNA is dephosphorylated, preventing its recognition by pattern recognition receptors on macrophages.
Add a cationic lipid-based transfection reagent to the mRNA, facilitating encapsulation of the mRNA through electrostatic interactions and forming a complex.
Introduce the lipid-mRNA complexes into a multi-well plate with adhered mouse-derived primary macrophages and gently agitate for uniform mixing.
The macrophages internalize the mRNA-containing complex into an endosome. Later, the complex fuses with the endosomal membrane, releasing the mRNAs into the cell cytoplasm.
The poly-A tail at the 3'-end stabilizes the mRNA which undergoes protein synthesis to produce green fluorescent proteins.
Under a fluorescence microscope, green fluorescence within the macrophages suggests the successful transfection with modified mRNAs.
Prepare for transfection by adding the pre-calculated volume of mRNA transfection buffer minus the volumes of mRNA transfection reagent and the mRNA to a reaction tube. Thaw the mRNA stock and mix it gently by flipping the tube. Then, add the pre-calculated volume of mRNA to the reaction tube.
Vortex and spin down the reaction mix and the mRNA transfection reagent, then add the appropriate volume of the mRNA transfection reagent to the reaction mix tube. Vortex the tube, and spin it down, then incubate it at room temperature for 15 minutes. Meanwhile, replace the culture medium of the macrophages with fresh, warm culture medium.
Once incubation of the transfection mix is complete, add the mix to the plate wells containing the macrophages dropwise and in a circle from the outside to the middle of the well. To ensure uniform distribution of the transfection mix, gently rock the plate in a vertical and horizontal direction.
Then incubate the plate at 37 degrees Celsius and 5% carbon dioxide for at least six hours. After the incubation, analyze transfection efficiency with fluorescence microscopy, flow cytometry, or immunoblot.
The most important part of this procedure is to ensure uniform distribution of the transfection mix so that all macrophages come in contact with the same amount of mRNA.
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