Preparation and Characterization of EGFP and Runx2 mRNA/pDNA

Enhanced GFP (EGFP) mRNA was purchased from Trilink (San Diego, CA, USA). Runt-related transcription factor 2 (Runx2) mRNA was prepared by in vitro transcription (IVT) as described previously [30 (link)]. Briefly, a flag-tagged mouse Runx2 DNA sequence, a kind gift from K. Miyazono (The University of Tokyo, Tokyo, Japan), was inserted into a pSP73 vector (Promega, Madison, WI, USA) possessing a 120 bp poly A/T sequence. IVT was performed using a mMESSAGE mMACHINE T7 ULTRA Kit (Ambion, Invitrogen, Carlsbad, CA, USA), followed by purification using an RNeasy mini kit (QIAGEN, Hilden, Germany) and spectroscopic measurement of concentration at 260 nm. Purity of mRNA was assessed spectroscopically based on ultraviolet (UV) absorption, and size of mRNA was evaluated by on-chip capillary electrophoresis using Bioanalyzer Agilent2100 (Agilent, Santa Clara, CA, USA). For pDNA construction, a sequence coding EGFP (Clontech, Palo Alto, CA, USA) and that coding a flag-tagged mouse Runx2 were inserted into pCAG-GS vectors (RIKEN, Tokyo, Japan). Notably, mRNA and pDNA were designed to possess the same protein coding sequences in both cases of EGFP (Genbank: JA532579.1) and Runx2 (RefSeq: NM_001145920.2).