Figure 1.

Production of Nb displaying LVs. (a) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene (y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs (x-axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown (n=6). (b) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean (n=6). (c) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown (n=3). (d) Western blot was performed as a quality control of the LVs. After separation on a 15% sodium dodecyl sulphate-polyacrylamide gel and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown (n=4). (e) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean (n=4).