Has anyone compared various transfection reagents comparing transfection efficiency across multiple cell lines...specifically for modified mRNAs? Also, I'm interested to know if anyone has evaluated innate immune response effects of various transfection reagents.
pei25b is appropriate for transfection and it has no effect on functional activity of Nf-kB one of the major signaling pathways in innate immune response.
Dear Mark,
I would definitively propose of course the RmesFect transfection reagent that shows very good efficiency for both cell lines and primary cells. Moreover an improved version was developped for stem cells.
So far, we never observed any innate immune response when using this reagent. All our transfection reagent are biodegradable and thus compatible with cell life, meaning that their impact on cells is minimized.
The important point in your post comes from the mRNA used. Depending on the mRNA, the immune response will be lowered or enhanced. For example it is well known that a non-modified mRNA will activate innate immune response when compared to a modified mRNA, but its stability will be lowered. Moreover, depending on the modification of the mRNA (not only the 5' Cap or 3' Tail), the immune response will be modulated.
So it is obvious that the transfection reagent's action on cells should be taken into account, but also the design and the mRNA.
I would be very happy to send you some sample of RmesFect ans RmesFect Stem; you can contact me via ResearchGate or directly at: [email protected]
For more information, you can refer to the link below,
do not hesitate to contact me,
best regards,
Cedric
http://www.ozbiosciences.com/transfection-rna/57-rmesfect-transfection-reagent-mrna-transfection.html
Thanks for the responses. Although PEI seems common for DNA, this paper indicates it doesn't work for mRNA: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55922/
Thanks for the RmesFect tip. I like the data on the Trilink website:
http://gallery.trilinkbiotech.com/tag/rmesfect/
and also the data from other manufacturers:
http://gallery.trilinkbiotech.com/tag/mrna-in/
Hey Matthias,
we struggled for a long time to transfect our macrophages. We had even more trouble because we use ex vivo peritoneal macrophages. They are very delicate and react nearly to everything. Viromer substances or Lipofectamine either did not work or killed the macrophages. RAW cells are easier to transfect (cancer-like cell line). However, we recently established a method for transfection with the JetMessenger transfection reagent 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 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 hope this was helpful to you. All the best, Marc
I know this is an old question but did anyone actually figure out what to use??
Regards
Mahmudul Hasan
Probably some of the RNA vaccine patents would give some good info, like this one: https://patents.google.com/patent/EP1619254A1/en
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