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A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles
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-...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8752749/ https://www.ncbi.nlm.nih.gov/pubmed/35017633 http://dx.doi.org/10.1038/s41598-021-04512-0 |
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author | Nørgård, Mikkel Ø. Steffensen, Lasse B. Hansen, Didde R. Füchtbauer, Ernst-Martin Engelund, Morten B. Dimke, Henrik Andersen, Ditte C. Svenningsen, Per |
author_facet | Nørgård, Mikkel Ø. Steffensen, Lasse B. Hansen, Didde R. Füchtbauer, Ernst-Martin Engelund, Morten B. Dimke, Henrik Andersen, Ditte C. Svenningsen, Per |
author_sort | Nørgård, Mikkel Ø. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8752749 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-87527492022-01-13 A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles Nørgård, Mikkel Ø. Steffensen, Lasse B. Hansen, Didde R. Füchtbauer, Ernst-Martin Engelund, Morten B. Dimke, Henrik Andersen, Ditte C. Svenningsen, Per Sci Rep Article 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. Nature Publishing Group UK 2022-01-11 /pmc/articles/PMC8752749/ /pubmed/35017633 http://dx.doi.org/10.1038/s41598-021-04512-0 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Nørgård, Mikkel Ø. Steffensen, Lasse B. Hansen, Didde R. Füchtbauer, Ernst-Martin Engelund, Morten B. Dimke, Henrik Andersen, Ditte C. Svenningsen, Per A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title | A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title_full | A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title_fullStr | A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title_full_unstemmed | A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title_short | A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles |
title_sort | new transgene mouse model using an extravesicular egfp tag enables affinity isolation of cell-specific extracellular vesicles |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8752749/ https://www.ncbi.nlm.nih.gov/pubmed/35017633 http://dx.doi.org/10.1038/s41598-021-04512-0 |
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