Reporter gene expression of systemically applied mCD105-LV. Mice were injected intravenously in the tail vein with mCD105-LV or VSV-G-LV (single dose of 2 × 106 transducing units, respectively), transferring a bicistronic luciferase-GFP reporter gene (pS-luc2-GFP-W). (A) In vivo luminescence imaging monitored 2 weeks after vector injection. Two representative mice out of 6 are shown for each group. (B) Mice were sacrificed at 2 weeks after vector injection, and livers were excised for histology. Images were acquired on a fully automated Axio-Observer Z1 microscope equipped with an ApoTome optical sectioning unit (Carl Zeiss, Jena, Germany). For quantification, 4 large high-resolution images from different liver areas were assembled by acquisition of 100 adjacent fields of view per section at 20× magnification using the MosaiX module (Carl Zeiss). The left panel shows a representative overview of mCD105-LV transduced mouse liver comprising 100 tiles at 20× magnification stained against CD31 (red), GFP (green), and with 4,6 diamidino-2-phenylindole (DAPI) (blue). Scale bar: 500 µm. The right panel shows a higher magnification of representative sections stained either against CD31 for LSEC (upper right) or against F4/80 for Kupffer cells (lower right). Scale bar: 25 µm. (C) Quantification of GFP colocalization with F4/80 or CD31, respectively, for mCD105-LV (red) and VSV-G-LV (blue) injected mice. Per marker and vector, 4 sections derived from 2 mice, corresponding to a total liver area of 62 mm2, in total more than 100 000 cells corresponding to about 500 transduction events, were analyzed. Morphometric analysis for F4/80/GFP-colocalization as well as nuclei quantification was performed using the Cell Profiler software.26 CD31/GFP colocalization was quantified by manual counting. (D) Results from morphometric analysis of Kupffer cell transduction. Intensities of GFP-positive cells were correlated to F4/80 staining and plotted against the total GFP intensity of the respective cells. Below the threshold of 0.35, no Kupffer cell transduction was observed.
