2021
DOI: 10.3390/ijms23010282
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CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

Abstract: Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the… Show more

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Cited by 9 publications
(20 citation statements)
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“…We used EVs from HEK293T cells engineered to express different versions of FP‐tagged CD63. This included stable, random integration of CD63‐mNeon (Wiklander et al., 2019 ) or CD63‐GFP (‘OE’, (Strohmeier et al., 2021 )), as well as transient transfection of CD63‐emGFP. In addition, we used a HEK293T line with GFP engineered into the CD63 locus by CRISPR/Cas9 technology (‘CD63‐GFP CRISPR’) (Strohmeier et al., 2021 ).…”
Section: Resultsmentioning
confidence: 99%
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“…We used EVs from HEK293T cells engineered to express different versions of FP‐tagged CD63. This included stable, random integration of CD63‐mNeon (Wiklander et al., 2019 ) or CD63‐GFP (‘OE’, (Strohmeier et al., 2021 )), as well as transient transfection of CD63‐emGFP. In addition, we used a HEK293T line with GFP engineered into the CD63 locus by CRISPR/Cas9 technology (‘CD63‐GFP CRISPR’) (Strohmeier et al., 2021 ).…”
Section: Resultsmentioning
confidence: 99%
“…This included stable, random integration of CD63‐mNeon (Wiklander et al., 2019 ) or CD63‐GFP (‘OE’, (Strohmeier et al., 2021 )), as well as transient transfection of CD63‐emGFP. In addition, we used a HEK293T line with GFP engineered into the CD63 locus by CRISPR/Cas9 technology (‘CD63‐GFP CRISPR’) (Strohmeier et al., 2021 ). EVs from these different parent cells were isolated by UF‐SEC (Supplementary File 3 ) and analysed by Fluorescence Correlation Spectroscopy (FCS) to determine the average number of fluorescent molecules per vesicle, following a previously described protocol (Corso et al., 2019 ).…”
Section: Resultsmentioning
confidence: 99%
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“…The uptake of these EVs was previously confirmed in HeLa cells; they remain mainly in the cytosol and in proximity to the nucleus . Nevertheless, no information on the uptake dynamics of EVs is yet available.…”
Section: Introductionmentioning
confidence: 87%