Transfer of luciferase-coding mRNA in vivo in mouse skin. For the experiments depicted in (a–h), mouse ear pinnae were injected with a fixed volume of 100 μl containing globin UTR-stabilized mRNA (RNActive) that encodes luciferase. The luciferase activity recovered from complete mouse ear lysate is indicated in amount of molecules on the y axis. The detection limit is 4–6 million luciferase molecules depending on the set of experiments and is symbolized by a line. Each dot indicates a single ear; the number of tested ears is written under the x axis after ‘N=’. Statistical significance between the experimental groups and the control group is shown in the upper part of each figure by a line ending on the P-value calculated according to a Mann–Whitney rank sum test. In (a, b, f, g and h), ears were removed 18 h after injection. In (a), different injection solutions were tested: PBS, Hepes/NaCl and Ringer lactate. In (b), the lack of lactate or calcium in Ringer was tested. In (c), the amount of luciferase molecules produced by human DCs electroporated in vitro in standard conditions (ca. 4 million of cells in 200 μl OptiMEM pulse at 300 V and 150 μF in a 0.4 cm cuvette) with 10 μg of luciferase-coding RNActive is shown: Luc stands for DCs transfected with luciferase-encoding RNActive and lacZ stands for DCs transfected with β-galactosidase-encoding RNActive. In (d), the expression of pDNA or RNActive vector (mRNA) was compared after injection of these nucleic acids dissolved in PBS or Ringer lactate, respectively. In (e), the localization of luciferase activity and the duration of the expression after pDNA (D) or RNActive (R) injection is reported using an in vivo imaging system. In (f), the effect of titrating the concentration of RNActive on luciferase activity was tested. In (g), an excess of several biomolecules (‘Comp’) was added to the RNActive to assess the capacity of these compounds to interfere with RNA uptake: irR stands for influenza matrix 1-coding RNActive, soR stands for luciferase-coding mRNA that is degraded by sonication, dsR stands for poly IC double-stranded RNA, dsD stands for double-stranded pDNA, ssD stands for single-stranded CpG phosphorothioate DNA oligonucleotide and Pep stands for peptide SYFPEITHI. In (h), several drugs were injected in mice before injection of RNActive: Amil stands for amiloride, Chpr stands for chloropromazine, Nyst stands for nystatin and CytB stands for cytochalasin B. DC, dendritic cells; mRNA, messenger RNA; PBS, phosphate-buffered saline; pDNA, plasmid DNA.