Construction of a mouse model that can be used for tissue-specific EV screening and tracing in vivo

Abstract

Extracellular vesicles (EVs) play an important role in the communication between tissues and cells. However, it is difficult to screen and trace EVs secreted by specific tissues in vivo, which affects the functional study of EVs in certain tissues under pathophysiological conditions. In this study, a Cre-dependent CD63flag-EGFP co-expressed with mCherry protein system expressing mice was constructed, which can be used for the secretion, movement, and sorting of EVs from specific tissues in vivo. This mouse model is an ideal research tool for studying the secretion amount, target tissue, and functional molecule screening of EVs in specific tissues under different pathophysiological conditions. Moreover, it provides a new research method to clarify the mechanism of secreted EVs in the pathogenesis of the disease.

Snorkel-tag Based Affinity Chromatography for Recombinant Extracellular Vesicle Purification

“…While tetraspanin-based reporters such as exomap1 68 and CD63-EGFP 69 generated transgenic animal models have been used previously, these reporter constructs contain the transgene inside of the EVs, thus lack the possibilities of using them as affinity isolation means, but most probably do not alter the surface characteristics of these EVs. On the other hand, CD63 FLAG -EGFP 70 and truncated CD9-EGFP 71 transgenic mouse models enable tracking and affinity purification of EVs, but the extracellular domains of the tetraspanins are altered, which can produce unintended biochemical changes in tetraspanins altering their functionality, post-translational modifications, subcellular localizations. Since the Snorkel-tag is placed after a 5th transmembrane domain distant from unaltered CD81 domains, it might be less likely to interfere with natural CD81 functionality but still is displayed to the outside, allowing not only tracking but also isolation to advance our knowledge on EV biogenesis, cell-type specific, physiologic and pathologic function and cargo in or ex vivo.…”

“…While tetraspanin-based reporters such as exomap1 68 and CD63-EGFP 69 generated transgenic animal models have been used previously, these reporter constructs contain the transgene inside of the EVs, thus lack the possibilities of using them as affinity isolation means, but most probably do not alter the surface characteristics of these EVs. On the other hand, CD63 FLAG -EGFP 70 and truncated CD9-EGFP 71…”

Exomap1 mouse: a transgenic model forin vivostudies of exosome biology

“…Although the exomap1 mouse was the first to be based on CD81 and the first to show the relative contributions of specific cell types to blood and CSF exosome population, it is not the first transgenic animal model to employ a highly-enriched exosome cargo protein, as previous studies have described animals that express CD9-GFP (97,98) or CD63-GFP (99)(100)(101)(102)(103). Of these, the TIGER mouse model is the most similar to the exomap1 mouse, as it carries a Cre-activated, CAG-regulated transgene designed to express a fusion between full-length human CD9 and GFP (HsCD9-GFP( 97)).…”

“…In addition to the CD81-based exomap1 and CD9-based TIGER mice, several groups have developed transgenic animals that express CD63 proteins tagged with a fluorescent protein (99)(100)(101)(102)(103). However, it is currently unclear whether these CD63-based models are the most appropriate tools for studying exosome biology in vivo, in part because CD63 is loaded into exosomes at ~15-fold lower efficiency than CD81 (5), in part because transgenic expression of CD63 is lethal (100), and in part because high-level expression of CD63 inhibits AP-2-mediated endocytosis, with complex effects on exosome content (44).…”