Studying extracellular vesicle transfer by a Cre-loxP method

TY - JOUR

T1 - Studying extracellular vesicle transfer by a Cre-loxP method

AU - Zomer, Anoek

AU - Steenbeek, Sander Christiaan

AU - Maynard, Carrie

AU - van Rheenen, Jacco

PY - 2016/1

Y1 - 2016/1

N2 - Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes ∼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼ 2 months.

AB - Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes ∼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to ∼ 2 months.

KW - Animals

KW - Attachment Sites, Microbiological

KW - Biological Transport

KW - Extracellular Vesicles

KW - Female

KW - Genetic Techniques

KW - HEK293 Cells

KW - Humans

KW - Integrases

KW - Mice

U2 - 10.1038/nprot.2015.138

DO - 10.1038/nprot.2015.138

M3 - Article

C2 - 26658469

SN - 1754-2189

VL - 11

SP - 87

EP - 101

JO - Nature Protocols

JF - Nature Protocols

IS - 1

ER -