Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity.
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.
Holder
Emma
E
Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
Stevenson
Barbara
B
Farley
Raymond
R
Hilliard
Tom
T
Wodehouse
Theresa
T
Somerton
Lucinda
L
Larsen
Mia
M
O'Donoghue
Jean
J
Coles
Rebecca L
RL
Scheule
Ronald K
RK
Cheng
Seng H
SH
Gill
Deborah R
DR
Hyde
Stephen C
SC
Griesenbach
Uta
U
Alton
Eric W F W
EW
Porteous
David J
DJ
Boyd
A Christopher
AC
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*
D050556:Fatty Acid-Binding Proteins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011402:Pronase