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Bioengineering targeting mRNA has been applied broadly in biology and biomedicine, including analysis of gene expression at the translational level, stem cell reprogramming, vaccination and substitutional therapy. Due to mRNA inability to integrate into the genome, mRNA transfection is considered as more direct and safe for therapeutic application than DNA-mediated techniques. In particular, the use of mRNA transfection for gene expression allows dissection of specific translational control mechanisms. This method does not require a delivery into the nucleus, simplifying the analysis of non-dividing cells. However, mRNA transfection of primary cells is tricky due to activation of the innate immune response. Introduction of modified nucleotides into a transcript can resolve this problem and also improve mRNA stability. However, published data on the effects of nucleotide modifications on protein products are controversial. Here, we thoroughly studied the effects of two modified bases, N1-methylpseudouridine and 5-methylcytidine, within a luciferase transcript, as well as three 5’ cap species, on reporter mRNA expression in various in vitro and in vivo systems. We compared translation efficiency and kinetics of regular and modified transcripts in cell extracts (commercially available rabbit reticulocyte lysate and an in-house prepared extract of mouse Krebs-2 ascite cells), in immortalized cultured cells (hepatocarcinoma Huh7, mouse fibroblasts NIH3T3 and human embryonic kidney cells HEK293T), and in primary mouse hepatocytes and fibroblasts. All mRNAs having m7G-cap efficiently produced luciferase for 1-2 hours (in the cell-free systems) and for up to 10-12 hours in living cells, but the effects of mRNA modifications on translation efficiency differed for the systems we used. In primary cells, the most efficient translation was directed by mRNAs transcribed in the presence of 50% N1-methylpseudouridine and 50% 5-methylcytidine, that had cap-0 structure at their 5’-termini. Our results can be used for the development of efficient mRNA transfection methods for transgene production in living animals. The work was supported by the grant of the Russian Federation government № 14.W03.31.0012