Synthesis of Modified mRNA with FLAG or EGFP

Murine Fam64a-FLAG or EGFP sequence downstream of T7 promoter was cloned into pGEMHE, and was PCR amplified using a primer set (3′-GTAAAACGACGGCCAGT-5' and 3′-CAGGAAACAGCTATGAC-5′). This PCR product was purified using FastGene Gel/PCR Extraction Kit (Nippon Genetics, Japan), and was used as the template for modified mRNA synthesis. In vitro transcription was performed using HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, USA) with a customized ribonucleoside blend of GTP (1.5 mM), ATP (7.5 mM), CTP (7.5mM), N1-Methylpseudo-UTP (7.5 mM, N-1081, Tri-Link Biotechnologies), and CleanCap® Reagent AG (6 mM, N-7113, Tri-Link Biotechnologies, USA) (Zangi et al., 2017 (link); Kaur and Zangi, 2020 (link)). Following the purification of transcribed mRNA using Fast Gene RNA Premium Kit (Nippon Genetics), Poly (A) tailing reaction was performed using E. coli Poly (A) polymerase (New England Biolabs), and mRNA was re-purified with the same kit. The size and the integrity of synthesized modified mRNA was checked by agarose gel electrophoresis, and quantity was determined using a NanoDrop One spectrophotometer (Thermo Scientific).