XPack-GFP Exosome Packing

  

The XPack-GFP fusion protein is efficiently packaged as exosome cargo and delivered to target cells. HEK-293 cells were transfected with a plasmid encoding an N-terminal XPack-GFP fusion protein (catalog # XPAK530PA-1). 24 hours later, GFP localization patterns were visualized on a Leica DMI300B fluorescent microscope (upper right panel). GFP expression patterns within transfected cells demonstrate a localization pattern characteristic of exosomal loading, with punctate spots along the cell periphery indicating budding vesicles and a brightly fluorescent spot in the cell interior corresponding to the multivesicular body (MVB) where exosomes are assembled and matured before secretion.

After 48 hours, exosomes were isolated using ExoQuick-T® and analyzed by Western blot for GFP loading (lower right panel). Equal amounts of exosomal lysates (15 ug) were separated on gradient SDS PAGE gels and detected for GFP protein expression. The control for exosome loading was an anti-CETP antibody. As shown in the image, XPack-GFP exosomes had a very high abundance of GFP protein loaded as cargo.

XPack-GFP Exosome Delivery to Target Recipient Cells

The XPack-GFP exosomes isolated from transfected cells described above were then added to recipient HEK-293 cells to test for GFP delivery. The cells were first imaged by fluorescent microscopy to assay uptake of XPack-GFP proteins (lower left panels). The patterns were consistent with endocytosis of the exosomes and delivery of the XPack-GFP protein cargo. Subsequently, these recipient cells were washed twice with PBS and cellular lysates were prepared for Western blot analysis probed with anti-GFP antibodies. Anti-GAPDH was used as a cellular protein loading control. The Western blot data further verified the successful delivery of GFP proteins to recipient cells via the XPack exosome engineering technology (lower right panels).