Year-end Sale % OFF 
Abstract
A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway.
Highlights
A family of water channels has been identified that facilitate water permeability in a variety of tissues
When 293 cells were infected with crude viral lysate (CVL), AQP5 protein could be readily detected in crude membranes
When 293 cells were infected with CVL, Northern blot analysis revealed a hybridization-positive band of ϳ1.6 kilobases
Summary
Cell Culture—Human 293 cells were grown in Eagle’s minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1,000 units/ml penicillin and 100 g/ml streptomycin and routinely passaged every 2–3 days using citric saline (Biofluids, Rockville, MD). MDCK cells (a gift of Dr Jared Grantham, University of Kansas, Kansas City, KS) were grown in Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1) medium containing 15 mM Hepes, pH 7.1–7.2, supplemented with 5% fetal bovine serum, 2 mM glutamine, 1,000 units/ml penicillin, and 100 g/ml streptomycin and routinely passaged once a week using trypsin. The cells were frozen and thawed three times and centrifuged at 5,000 ϫ g for 10 min, and the crude viral lysate (CVL), containing the recombinant adenovirus (AdrAQP5), was used to infect confluent 293 cells. The presence of -galactosidase (the LacZ gene product) in Ad.RSVGal-infected MDCK cells was determined after 3 days using the X-Gal substrate as described (13). Antibodies 2 and 3 gave immunopositive results on MDCK cells
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
