Methods for Lymphatic Vessel Culture and Gene Transfection
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OBJECTIVE: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself. METHODS: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3-12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells. RESULTS: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3-12 days without significant impairment of the parameters of their pumping and effective adenoviral/GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. CONCLUSIONS: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.
author list (cited authors)
Gashev, A. A., Davis, M. J., Gasheva, O. Y., Nepiushchikh, Z. V., Wang, W., Dougherty, P., ... Zawieja, D. C.