Premium
Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity
Author(s) -
Holder Emma,
Stevenson Barbara,
Farley Raymond,
Hilliard Tom,
Wodehouse Theresa,
Somerton Lucinda,
Larsen Mia,
O'Donoghue Jean,
Coles Rebecca L.,
Scheule Ronald K.,
Cheng Seng H.,
Gill Deborah R.,
Hyde Stephen C.,
Griesenbach Uta,
Alton Eric W. F. W.,
Porteous David J.,
Boyd A. Christopher
Publication year - 2010
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1413
Subject(s) - taqman , respiratory epithelium , laser capture microdissection , microbiology and biotechnology , microdissection , transfection , gene expression , real time polymerase chain reaction , transgene , biology , epithelium , mucous membrane of nose , nose , pronase , gene , immunology , biochemistry , enzyme , anatomy , genetics , trypsin
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. Copyright © 2009 John Wiley & Sons, Ltd.