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Team:MIT/GFP
From 2013.igem.org
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Revision as of 03:08, 28 September 2013
Overview
To visualize the localization and exosomal exportation of Acyl-TyA, the enhanced green fluorescent protein (eGFP) was added to the C-terminus and the expression of the fusion protein (Acyl-TyA-eGFP) was driven by a human cytomegalovirus (CMV) promoter.
Method:
Previous research on biogenesis of exosomes has shown the rhodamine phosphatidylethanolamine (Rh-PE) preferentially labels the endosome-like-domain (ELD), which is believed to be the exosomal budding site on cell membrane. 24 hours after Jurkat T cells were nucleofected with CMV_Acyl-TyA-eGFP construction, the cells were stained with Rh-PE. The colocalization of the stain and green fluorescence was observed by confocal for 24 hrs.
Results:
Rh-PE was seen to localize at the ELD immediately after staining procedure. Over the course of 24 hrs, exosomes budded out with stain. 48 hrs post nucleofection, the green fluorescence was observed to localize on cell membrane.
