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.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

“…A key strength of in vivo models is the opportunity to assess the release of physiological levels of EVs and their interaction with target cells. Some in vivo studies examine endogenous EVs, usually using fluorescent (Estrada et al, 2022;Hegyesi et al, 2022;Neckles et al, 2019;Nørgård et al, 2022) or bioluminescent tags (Gupta et al, 2020;Luo et al, 2020;Rufino-Ramos et al, 2022). Preclinical studies with syngeneic models and human cancer cell line xenograft models have allowed tumour and other EVs to be specifically labelled and traced (Driedonks et al, 2022;Hyenne et al, 2019;Liu et al, 2016;Pucci et al, 2016;Wiklander et al, 2015).…”

Extracellular vesicles in kidney disease

“…Crisp-Cas9 technology has also been exploited to generate functional reporters for tissue localization of extracellular vesicles. A transgenic reporter mouse that used Cre-dependent extracellular vesicle labelling under the renal promoter Pax8 enabled the detection of tubular epithelial cell-derived extracellular vesicles in urine 133 . This strategy provides a new approach to studying cell-specific extracellular vesicles in vivo.…”

Using human urinary extracellular vesicles to study physiological and pathophysiological states and regulation of the sodium chloride cotransporter

“…or systemic EVs can also be detected in urine ( 41 , 42 ), suggesting that EVs can cross the glomerular filtration barrier and basement membrane of the kidney. How this occurs is unclear, but recent studies in transgenic mice have emphasized that under physiological conditions the majority of uEVs are derived from the kidney with limited contribution of EVs from the circulation ( 43 ). In contrast, in pathophysiological states this contribution may be different.…”