title: Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity.
abstract: BACKGROUND
  When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs).
  
  
  METHODS
  We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model.
  
  
  RESULTS
  Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs (n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs (n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels.
  
  
  CONCLUSIONS
  Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy.
authors: Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
authors: Emma
authors: E
authors: Holder
authors: Barbara
authors: B
authors: Stevenson
authors: Raymond
authors: R
authors: Farley
authors: Tom
authors: T
authors: Hilliard
authors: Theresa
authors: T
authors: Wodehouse
authors: Lucinda
authors: L
authors: Somerton
authors: Mia
authors: M
authors: Larsen
authors: Jean
authors: J
authors: O'Donoghue
authors: Rebecca L
authors: RL
authors: Coles
authors: Ronald K
authors: RK
authors: Scheule
authors: Seng H
authors: SH
authors: Cheng
authors: Deborah R
authors: DR
authors: Gill
authors: Stephen C
authors: SC
authors: Hyde
authors: Uta
authors: U
authors: Griesenbach
authors: Eric W F W
authors: EW
authors: Alton
authors: David J
authors: DJ
authors: Porteous
authors: A Christopher
authors: AC
authors: Boyd
mesh_terms: D000818:Animals; D002469:Cell Separation; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection; D009296:Nasal Cavity / Q000378:metabolism / Q000473:pathology*; D009300:Nasal Septum / Q000378:metabolism / Q000473:pathology; D011402:Pronase / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology*; D019076:Transgenes / Q000235:genetics*
chemical_list: D050556:Fatty Acid-Binding Proteins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011402:Pronase