The pDNA and mRNA were based on the same construction process as previously reported [6 (link),7 (link)].
pNL1.3 CMV-encoding secreted NanoLuc (secNluc pDNA) for in vitro experiments and pGL4.51 [luc2/CMV/Neo] encoding Luc2 (Luc2 pDNA) for in vivo experiments were obtained from Promega (Madison, WI, USA). Amplification of pDNA was performed in Escherichia coli DH5α strain. After isolation, the pDNA was purified using an endotoxin-free plasmid purification kit. The resulting pDNA was dissolved in Milli-Q water and stored at −20 °C until use in each experiment.
DNA templates for in vitro transcription (IVT) of mRNA encoding Gluc (Gluc mRNA) for in vitro experiments and Luc2 (Luc2 mRNA) for in vivo experiments were prepared by inserting protein expression fragments into a pSP73 vector (Promega, Madison, WI, USA) containing the T7 promoter. Prior to insertion, a 120 bp poly A/T sequence was introduced downstream of the protein-coding sequence in the pSP73 vector. This modification allowed us to produce mRNA with a 120 adenine poly(A) tail at the 3’ end by a simple IVT procedure using the pSP73-poly(A) vector. The protein expression fragment was derived from DNA encoding firefly luciferase (pGL4; Promega, Madison, WI, USA).
pNL1.3 CMV-encoding secreted NanoLuc (secNluc pDNA) for in vitro experiments and pGL4.51 [luc2/CMV/Neo] encoding Luc2 (Luc2 pDNA) for in vivo experiments were obtained from Promega (Madison, WI, USA). Amplification of pDNA was performed in Escherichia coli DH5α strain. After isolation, the pDNA was purified using an endotoxin-free plasmid purification kit. The resulting pDNA was dissolved in Milli-Q water and stored at −20 °C until use in each experiment.
DNA templates for in vitro transcription (IVT) of mRNA encoding Gluc (Gluc mRNA) for in vitro experiments and Luc2 (Luc2 mRNA) for in vivo experiments were prepared by inserting protein expression fragments into a pSP73 vector (Promega, Madison, WI, USA) containing the T7 promoter. Prior to insertion, a 120 bp poly A/T sequence was introduced downstream of the protein-coding sequence in the pSP73 vector. This modification allowed us to produce mRNA with a 120 adenine poly(A) tail at the 3’ end by a simple IVT procedure using the pSP73-poly(A) vector. The protein expression fragment was derived from DNA encoding firefly luciferase (pGL4; Promega, Madison, WI, USA).
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