Construction and function of a recombinant adenovirus encoding a human aquaporin 1-green fluorescent protein fusion product.
Transfer of the human aquaporin 1 (hAQP1) gene provides a novel way to potentially correct the severe salivary hypofunction associated with therapeutic radiation for head and neck cancer. To facilitate the study of individual cells transduced with this gene, we have designed a fusion product of the hAQP1 and jellyfish green fluorescent protein (GFP) cDNAs. An expression plasmid, pACCMVhAQP1GFP, and a recombinant adenovirus, AdhAQP1GFP, encoding this fusion product were constructed. Both the recombinant plasmid and virus directed the expression of the encoded, 55-kDa fusion protein (hAQP1GFP), which was detected in the plasma membranes of several epithelial cell lines (293, SMIE, and A5). hAQP1GFP was functionally active and facilitated fluid movement across a polarized salivary epithelial cell monolayer (approximately 5-fold noninfected controls) in response to an osmotic gradient. In response to a hypotonic challenge, individual epithelial cells expressing the fusion protein exhibited significantly more capacitance (used herein as an indicator of cell swelling) than control cells. Conversely, in response to a hypertonic challenge, individual infected cells shrunk more rapidly (approximately 2- to 3-fold) and to a greater extent than control cells. We conclude that AdhAQP1GFP is a useful experimental tool to identify and study individual cells expressing a water channel transgene.
Hoque
A T
AT
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Liu
X
X
Kagami
H
H
Swaim
W D
WD
Wellner
R B
RB
O'Connell
B C
BC
Ambudkar
I S
IS
Baum
B J
BJ
D000256:Adenoviridae / Q000235:genetics*; D051398:Aquaporin 1; D020346:Aquaporins / Q000032:analysis / Q000235:genetics*; D001789:Blood Group Antigens; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000821:virology; D002463:Cell Membrane Permeability / Q000235:genetics; D004847:Epithelial Cells / Q000502:physiology / Q000821:virology; D005822:Genetic Vectors / Q000138:chemical synthesis; D049452:Green Fluorescent Proteins; D006801:Humans; D007424:Intracellular Fluid / Q000502:physiology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D010948:Viral Plaque Assay
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051398:Aquaporin 1; D049452:Green Fluorescent Proteins