Figure 4.

Transfection efficiency and EGFP mRNA expression level in macrophages transfected with IVT-mRNA with various cap and nucleotide modifications

(A) Representative fluorescent images and (B) flow cytometric density plots indicating EGFP expression in macrophages transfected with low dose and high dose of IVT-mRNA made of either Cap 0 (i.e., ARCA), or Cap 1 (i.e., ARCA + MT, and CleanCap) with and without phosphatase treatment. (C) Quantification of transfection efficiency, and (D) EGFP mRNA expression level in macrophages transfected with low doses and high doses of the different IVT-mRNAs with cap modifications. (E) Representative fluorescent images of macrophages transfected with low doses of IVT-mRNA composed of nucleotides with different chemical modifications. (F) Flow cytometric density plots indicating EGFP expression in macrophages transfected with low dose and high dose of IVT-mRNA with various nucleotide modifications. (G) Transfection efficiency and (H) EGFP mRNA expression level quantified by flow cytometry and plotted in terms of EGFP-positive cells percentage and MFI of EGFP signal among EGFP-positive cell populations, respectively. Poly(I:C) was also transfected in low dose (125 ng∙mL⁻¹). For each condition 125 ng∙mL⁻¹ and 500 ng∙mL⁻¹ of IVT-mRNA were used for transfection referred here as low dose and high dose, respectively. Values are presented as mean ± SD. Error bars indicate SD of three independent experiments from three individual donors. Bar = 50 μm. Statistical differences are depicted with ∗∗p < 0.005, ∗∗∗p < 0.001. ARCA, anti-reverse cap analog; MT, methyl-transferase; AnP, Antarctic phosphatase; Ψ, pseudouridine; me¹Ψ, N¹-Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methyl-cytidine.