| Popis: |
The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate. We therefore created an EV reporter using CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9-EGFP expression in cells did not affect EV size and concentration, but allowed for co-precipitation of EV markers TSG101 and ALIX from cell-conditioned medium by anti-GFP immunoprecipitation. We created a transgenic mouse where CD9-EGFP was inserted in the inverse orientation and double-floxed, ensuring Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV- Cre), in cardiomyocytes (AMHC-Cre) and kidney epithelium (Pax8-Cre), respectively. The mice showed tissue-specific EGFP expression, and plasma and urine samples were used to immunoprecipitate EVs. CD9-EGFP EVs was detected in plasma samples from CMV-Cre/CD9-EGFP and AMHC-Cre/CD9-EGFP mice, but not in PAX8-Cre/CD9-EGFP mice. On the other hand, CD9-EGFP EVs were detected in urine samples from CMV-Cre/CD9-EGFP and PAX8-Cre/CD9-EGFP mice, but not AMHC-Cre/CD9-EGFP, indicating that plasma EVs are not filtered to the urine. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to study cell-specific EVs in vivo and gain new insight into their physiological and pathophysiological function. |
