Figure 3.

eExos express packaged plasmids in vitro and in vivo. (A) Schematic of experiment: Transfection of HEK293T cells with plasmids encoding Gag, VSVg, and Cre results in generation of eExos containing pCAG-Cre. These eExos were applied to a U87 cell line that stably expressed the Cre-LoxP-DsRed/eGFP reporter; the reporter was introduced using lentiviral delivery. These cells fluoresce red in the absence of Cre. Expression of Cre protein from pCAG-Cre results in color change from red to green in U87 cells. Created with BioRender.com (B) HEK293T cells were transfected with pCre and Gag and/or VSVg. eExos were isolated and added to U87dsRed/GFP cells (10³ exos/cell). As a positive control, the same number of lentiviral particles encoding Cre (LV PuroCre(Gag/VSVg)) were used for comparison. After 72 hours, pictures were taken to observe the color change. (C) FACS analysis was conducted to quantify percentage of cells that changed color and FACS results are shown as a graph. (D) Transcription of Cre mRNA. After 48 hours, U87 dsRed/GFP recipient cells were harvested and RNA was isolated, and reverse-transcribed to cDNA. Reverse transcriptase PCR was conducted for Cre and GAPDH. (E) Western blot for Cre and a-tubulin of protein isolated from U87 dsRed/GFP cells 96 hours after they received exosomes. (F) Packaging limit of eExos(Gag/VSVg) using 6.8-kb, 8.1-kb, and 10-kb Cre pDNA. The functional packaging limit is 8.1 kb. Data shown are representative of >3 separate experiments.