A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Mikkel Nørgård; Lasse B. Steffensen; Didde R. Hansen; Ernst Martin Füchtbauer; Morten B. Engelund; Henrik Dimke; Ditte C. Andersen; Per Svenningsen

doi:10.1038/s41598-021-04512-0

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

Mikkel Nørgård, Lasse B. Steffensen, Didde R. Hansen, Ernst Martin Füchtbauer, Morten B. Engelund, Henrik Dimke, Ditte C. Andersen, Per Svenningsen^*

^*Corresponding author for this work

Department of Molecular Biology and Genetics - Cellular Health, Intervention, and Nutrition

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Original language	English
Article number	496
Journal	Scientific Reports
Volume	12
ISSN	2045-2322
DOIs	https://doi.org/10.1038/s41598-021-04512-0
Publication status	Published - Jan 2022

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title = "A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles",

abstract = "The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.",

author = "Mikkel N{\o}rg{\aa}rd and Steffensen, {Lasse B.} and Hansen, {Didde R.} and F{\"u}chtbauer, {Ernst Martin} and Engelund, {Morten B.} and Henrik Dimke and Andersen, {Ditte C.} and Per Svenningsen",

note = "Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = jan,

doi = "10.1038/s41598-021-04512-0",

language = "English",

volume = "12",

journal = "Scientific Reports",

issn = "2045-2322",

publisher = "Nature Publishing Group",

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A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles. / Nørgård, Mikkel; Steffensen, Lasse B.; Hansen, Didde R. et al.
In: Scientific Reports, Vol. 12, 496, 01.2022.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

AU - Nørgård, Mikkel

AU - Steffensen, Lasse B.

AU - Hansen, Didde R.

AU - Füchtbauer, Ernst Martin

AU - Engelund, Morten B.

AU - Dimke, Henrik

AU - Andersen, Ditte C.

AU - Svenningsen, Per

PY - 2022/1

Y1 - 2022/1

N2 - The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

AB - The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

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U2 - 10.1038/s41598-021-04512-0

DO - 10.1038/s41598-021-04512-0

M3 - Journal article

C2 - 35017633

AN - SCOPUS:85122785766

SN - 2045-2322

VL - 12

JO - Scientific Reports

JF - Scientific Reports

M1 - 496

ER -

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

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