When transfecting with mRNA, does the cell package the foreign RNA into vesicles reducing the efficiency of the mRNA, or are they left in the cytoplasm? If packaged away, what percent is packaged vs. what percent is left to be available for transcription? Is this observed with DNA plasmids?

Answer

We have not observed differences between how a cell packages an mRNA payload versus a DNA payload for the purpose of delivery.  Transfection involves complexation formation between a liposome and mRNA, which create lipoplexes that are taken up by the cell via endocytosis.  The liposome protects the mRNA during this process and also assists in endosomal escape, which releases the mRNA into the cytoplasm of the cell.  The mRNA is immediately available for translation with the ribosome.  The mRNA itself should be prepared using an in vitro transcription kit, such as mMessage mMachine® T7 Ultra Kit, which incorporate a 5’ ARCA cap and a 3’ poly(a) tail so that the mRNA mimics endogenous mRNA.