We are trying to transiently express a CFP-tagged protein in primary mouse bone marrow derived macrophages. Before we try jetOPTIMUS® or jetPEI®-Macrophage from Polyplus we would like to know how well they work.
Hey Carl,
we struggled for a long time to transfect our macrophages. We had even more trouble because we use ex vivo peritoneal macrophages. But also with BMDM it took a while until we got them transfected.
Viromer substances or Lipofectamine either did not work or killed the macrophages. Moreover, every attempt to transfect DNA led to macrophage death and nothing was expressed. However, we recently established a method for transfection with the JetMessenger transfection reagent from Polyplus and in vitro transcribed and immunologically silenced mRNA. Modifications during mRNA transcribtion were:
Removing any traces of DNA templates (because this induces macrophage death).
Use of 5-methyl-CTP and pseudo-UTP, which are bases that reduce immunogenicity and enhance stability of the mRNA.
Dephosphorylation of the 5’-ends of the in vitro transcribed mRNA by Antarctic phosphatase to prevent recognition by the RIG-I complex.
All of these steps together led finally to sucessful macrophage transfection, but it took long to get it right. For a detailed protocol (including GFP as an example) please have a look at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).
It worked for peritoneal macrophages, BMDM and MEFS.
But it only delivers mRNA and not plasmids. Tissues macropages go instantly into apoptosis, when we tried to transfect DNA. I am always a little sceptic, when people manage to transfect DNA into a macrophage (RAWs a more cancer cell then macrophage) because, as an immune cell, exogenous or endogenous DNA should not be in the cytosol and macrophages react with a lot of side effects.
I hope this was helpful to you.
All the best,
Marc
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