Genetic labeling of extracellular vesicles for studying biogenesis and uptake in living mammalian cells.

Autor:	Levy D; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Do MA; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Brown A; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Asano K; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Diebold D; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Chen H; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Zhang J; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Lawler B; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States., Lu B; Department of Bioengineering, Santa Clara University, Santa Clara, CA, United States. Electronic address: blu2@scu.edu.
Jazyk:	angličtina
Zdroj:	Methods in enzymology [Methods Enzymol] 2020; Vol. 645, pp. 1-14. Date of Electronic Publication: 2020 Feb 26.
DOI:	10.1016/bs.mie.2020.02.001
Abstrakt:	Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine. (© 2020 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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