|
Author
Sánchez Gómez, Maria José
|
Abstract
The development of new techniques for the diagnosis and treatment of multiple pathologies has been a challenge in recent years. Exosomes have recently emerged as new delivery systems due to their attractive natural origin, their nano-scale size, and their intrinsic role in intercellular communication. However, there are several challenges that prevent their use as therapeutic systems. Among them, the distinction of exosomes from the rest of the components (other vesicles, proteins, and extracellular particles), with a minimal alteration of their physical-chemical and biological properties, continues to be a key issue. Other drawbacks that still need to be overcome are the difficulties associated with exosome purification, absorption study, and biodistribution.
In this work, the selective labeling of exosomes with specific markers was carried out in A549 and HEK293 cell lines, which allowed their specific monitoring in addition to other vesicles and extracellular particles. For this, several plasmids were constructed to express CD63 and CD69, two proteins of the tetraspanin family located specifically in the membranes of exosomes, fused with fluorescent EGFP or mCherry tags. Furthermore, bicistronic and tricistronic plasmids were constructed to allow the simultaneous expression of one or two genes together with an antibiotic-resistant selection marker, allowing the efficient generation and selection of stable cell lines. The expression of all the fusion proteins designed in this work was analyzed by flow cytometry together with visualization of fluorescent and confocal microscopy. Furthermore, a stable culture of fluorescence-expressing HEK293 cells was achieved by mammalian antibiotic resistance combined with sorting technology.
In conclusion, the desired plasmid cloning was achieved and shown to be fully functional. The expression of the plasmids studied in this project makes fluorescent labeling of vesicles possible. This labeling is an advantage for future research related to exosome isolation, biodistribution, and its potential functions as a promising therapeutic vehicle.
|
|
