title: An immunocytochemical assay to detect human CFTR expression following gene transfer.
abstract: BACKGROUND
  To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.
  
  
  METHODS
  We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR.
  
  
  RESULTS
  The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells.
  
  
  CONCLUSIONS
  We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.
authors.affiliation: Medical Genetics, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH42XU, UK. H.Davidson@ed.ac.uk
authors.forename: Heather
authors.initials: H
authors.lastname: Davidson
authors.forename: Abigail
authors.initials: A
authors.lastname: Wilson
authors.forename: Robert D
authors.initials: RD
authors.lastname: Gray
authors.forename: Alex
authors.initials: A
authors.lastname: Horsley
authors.forename: Ian A
authors.initials: IA
authors.lastname: Pringle
authors.forename: Gerry
authors.initials: G
authors.lastname: McLachlan
authors.forename: Angus C
authors.initials: AC
authors.lastname: Nairn
authors.forename: Cordelia
authors.initials: C
authors.lastname: Stearns
authors.forename: James
authors.initials: J
authors.lastname: Gibson
authors.forename: Emma
authors.initials: E
authors.lastname: Holder
authors.forename: Lisa
authors.initials: L
authors.lastname: Jones
authors.forename: Ann
authors.initials: A
authors.lastname: Doherty
authors.forename: Rebecca
authors.initials: R
authors.lastname: Coles
authors.forename: Stephanie G
authors.initials: SG
authors.lastname: Sumner-Jones
authors.forename: Marguerite
authors.initials: M
authors.lastname: Wasowicz
authors.forename: Michelle
authors.initials: M
authors.lastname: Manvell
authors.forename: Uta
authors.initials: U
authors.lastname: Griesenbach
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Hyde
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Gill
authors.forename: Jane
authors.initials: J
authors.lastname: Davies
authors.forename: D David S
authors.initials: DD
authors.lastname: Collie
authors.forename: Eric W F W
authors.initials: EW
authors.lastname: Alton
authors.forename: David J
authors.initials: DJ
authors.lastname: Porteous
authors.forename: A Christopher
authors.initials: AC
authors.lastname: Boyd
mesh_terms: D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008856:Microscopy, Fluorescence; D009297:Nasal Mucosa / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000378:metabolism / Q000473:pathology; D014162:Transfection; D019076:Transgenes
chemical_list: D000906:Antibodies; C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins