electronic chiming Because macrophages specialize in detecting molecules of non-self origin, they are particularly hard to transfect. We describe a protocol which allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids. The main advantage of our method is that high transfection rates are achieved in the absence of any detectable cytotoxicity or immunogenicity.
Demonstrating the procedure will be Marc Herb a postdoc from my laboratory. Begin by thawing all components of the RNA transcription kit. Vortex the reagents for five seconds and spin them down for two seconds at 2, 000 times g, then keep them on ice until use.
Prepare the in vitro RNA transcription reaction in a 0.5-microliter microcentrifuge tube according to the manuscript directions. Vortex the tube and spin it down. Then incubate it at 37 degrees Celsius for 30 minutes while mixing at 400 rpm on a thermal mixer.
After the incubation, add two microliters of DNase I directly into the reaction mix. Vortex and spin the tube down, and incubate it in the thermal mixer for another 15 minutes. Reserve a two-microliter aliquot of the Dnase-treated product for the control gel and store it at minus 80 degrees Celsius.
For polyA tailing, add five microliters of 10X polyA polymerase reaction buffer and five microliters of polyA polymerase to the Dnase-treated reaction. Vortex and spin down the mix. Then incubate it in the thermal mixer for 30 minutes.
When the reaction is complete, take a two-microliter aliquot for the control gel and store it at minus 80 degrees Celsius. Add 5-prime dephosphorylation reagents directly to the polyA tailed product according to the manuscript directions. Vortex and spin down the tube, and incubate it in the thermal mixer for another 30 minutes.
Follow the dephosphorylation reaction with a two-minute incubation at 80 degrees Celsius in order to heat inactivate the Antarctic phosphatase. Then purify the in vitro transcribed mRNA using a dedicated RNA purification kit. Measure the concentration and purity of the eluted mRNA with a microvolume spectrophotometer.
Run a denaturing agarose gel with the previously collected aliquots to verify the presence of a single product, correct transcript length, and polyA tailing. 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 get in contact with the same amount of mRNA. This protocol was successfully used to generate mRNA encoding FLAG-tagged NEMO and IKK-beta variants for transfection of primary macrophages. Correct size and polyA tailing of the mRNA was verified by agarose gel electrophoresis.
Transfection efficiency was confirmed by generating mRNA encoding GFP and performing flow cytometry analysis. Transfection rate increased along with the amount of transfected mRNA, reaching 50 to 65%for PM and 80 to 85%for BMDM. In both PM and BMDM, expression level of EGFP in transfected cells increased in a time and dose-dependent manner.
Importantly, analysis of propidium iodide-positive or Annexin V-positive macrophages confirmed that the transfection procedure did not induce lytic or apoptotic cell death. Transfection efficiency of mRNAs encoding FLAG-tagged Nemo or IKK-beta variants were analyzed with immunofluorescence microscopy. The rates were about 60%for Nemo mRNAs and 55%for IKK-beta mRNAs.
This protocol was also used to generate mRNA encoding Cre recombinase. Transfection of 400, 000 BMDM with 400 nanograms of Cre recombinase mRNA resulted in almost complete depletion of Nemo protein after 48 hours, indicating highly efficient transfection. The absence of any detectable amounts of Interleukin 1 beta, Interleukin-6 and tumor necrosis factor indicate that the transfection procedure does not activate pro-inflammatory signaling.
Our relatively simple method finally allows to efficiently express mutated or tagged proteins in primary macrophages. This would substantially further the analysis of macrophage functions on the molecular level.
