title: Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.
abstract: Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed β5-GFP after liposome-mediated transfection. The percentage of cells with detectable β5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing β5-GFP but increased variability of β5-GFP level among cells. In cells with low expression levels, β5-GFP localized mostly to the apical plasma membrane like endogenous αvβ5 integrin. In cells with high expression levels, β5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture.
authors.affiliation: Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Larkin Hall, 441 East Fordham Road, 10458, Bronx, NY, USA. cmuller3@fordham.edu.
authors.forename: Claudia
authors.initials: C
authors.lastname: Müller
authors.affiliation: Department of Development and Regenerative Biology, Icahn School of Medicine at Mount Sinai, 1425 Madison Ave, Icahn Medical Institute, 10029, New York, NY, USA. timothy.blenkinsop@mssm.edu.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Blenkinsop
authors.affiliation: Neural Stem Cell Institute, 12144, Rensselaer, NY, USA.
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stern
authors.affiliation: Department of Biological Sciences, Center for Cancer, Genetic Diseases and Gene Regulation, Fordham University, Larkin Hall, 441 East Fordham Road, 10458, Bronx, NY, USA. finnemann@fordham.edu.
authors.forename: Silvia C
authors.initials: SC
authors.lastname: Finnemann
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D016764:Cell Polarity; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D039641:Integrin beta Chains / Q000235:genetics / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods
chemical_list: D039641:Integrin beta Chains; D008081:Liposomes; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C084344:integrin beta5; D049452:Green Fluorescent Proteins
keywords: Infectivity; Primary human fetal RPE; Protein expression; RPE; RPESC-RPE; Recombinant adenovirus; β5 integrin-GFP