EV Shuttle Kit with HEK293 Exosomes

Supporting Data

See the EV Shuttle System deliver cargo and alter cellular physiology even in hard-to-transfect cells

The EV Shuttle System delivers functional siRNA
 

Figure 1. Mouse and Human EV Shuttles efficiently deliver siRNA to hard-to-transfect cells. (Left panel set) JAWS II mouse dendritic cell exosomes were incubated with Exo-Fect and 100 pmol Texas Red-conjugated siRNA (non-targeting control included in the EV Shuttle Kits). The siRNA-loaded EV Shuttles were then added to naive mouse monocyte macrophage cells (RAWS 264.7) in culture. The cells were imaged after 18 hours and delivery was observed as soon as 4 hours after adding the EV Shuttles. More than 80% of the recipient RAWS 264.7 cells internalized the EV Shuttle siRNA cargo.
(Right panel set) Human HEK 293 EV Shuttles were treated with SBI’s Exo-Green Kit (Cat.# EXOG200A-1) which fluorescently labels internal exosome proteins. The labeled Human EV Shuttles were then added to mouse embryonic stem cells and imaged for cargo delivery after 18 hours. The Human EV Shuttles were well-tolerated and the EVs were taken up efficiently by the mouse embryonic stem cells.

Figure 2. EV Shuttles are more efficient at delivering siRNA to hard-to-transfect cells than lipofectamine. Standard Lipofectamine transfection protocols were also compared to Human EV Shuttles on RAWS 264.7 cells using 100 pmol of Texas Red-labeled control siRNA. Fluorescence imaging shows more staining in the EV Shuttle-treated cells than the lipofectamine cells, demonstrating the higher transfection efficiency of the EV Shuttle System.

Figure 3. EV Shuttles deliver functional siRNA that can knockdown protein expression in recipient cells. Human HEK293 EV Shuttles were loaded with either an anti-CD81 siRNA or a non-targeting siRNA (NT) and then added to Mouse RAWS 264.7 cells. The cell lysates were collected after 18 hours for Western blot analysis of CD81 protein expression. EV Shuttles carrying the anti-CD81 siRNA were able to reduce CD81 protein expression in the target cells by at least 50% compared to EV Shuttles carrying the NT control siRNA.

EV Shuttles can be used as a non-viral method for generating stable cell lines

Because plasmids can be transfected into exosomes with the EV Shuttle System, EV Shuttles can be used to generate stable cell lines without the use of viruses. Here we show an example using our non-viral

Figure 4. EV Shuttles can be used to generate stable cell lines. The Super PB Transposase Expression Plasmid (5 µg) and 5 µg of the PB713B-1 PB Transposon Vector expressing GFP and a puromycin resistance gene were loaded into Human EV Shuttles and then added to mouse embryonic stem cells in culture. ES cells were placed under 0.5 µg/mL puromycin selection for 3 days, and then 1 µg/mL puromycin for an additional 4 days of selection. As a control, EV shuttles were loaded without the Super PB Transposase Vector. Fluorescence imaging shows the successful genomic integration of Human EV Shuttle-mediated PiggyBac transposons into mouse ES cells.

Figure 5. EV Shuttles are more efficient than lipofectamine. We again used the Super PB Transposase Expression Plasmid and the PB713B-1 PB Transposon Vector expressing GFP and a puromycin resistance gene loaded into Human EV Shuttles. Loaded EV Shuttles were added to naïve HEK293 cells, and placed under puromycin selection first at 2 µg/mL for 72 hours post EV Shuttle treatment, and then at 5 µg/mL for an additional 72 hours. EV Shuttle-mediated transpositioned HEK293 cells were imaged post selection (upper panels) and compared to cells that were Lipofectamine transfected with the same PB plasmids (lower panels).