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Published ahead of print on February 23, 2006, doi:10.1165/rcmb.2005-0377OC

Am. J. Respir. Cell Mol. Biol., Volume 35, Number 1, July 2006, 72-83

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Submitted on October 7, 2005
Revised on February 23, 2006

Human Specific CFTR Antibodies Detect in vivo Gene Transfer to Ovine Airways

Heather Davidson1*, Gerry McLachlan2, Abigail Wilson1, A. Christopher Boyd1, Ann Doherty1, Gordon MacGregor1, Lee Davies3, Hazel A Painter3, Rebecca Coles3, Stephen C Hyde3, Deborah R Gill3, Margarida D Amaral4, David D. S. Collie2, David J Porteous1, and Deborah Penque5

1 Department of Medical Sciences (Medical Genetics), University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom; UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom, 2 The Wellcome Trust Centre in Comparative Respiratory Medicine, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, United Kingdom; UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom, 3 Gene Medicine Group, Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom, 4 Centro de Genetica Humana, Instituto Nacional Saude Dr Ricardo Jorge, Lisboa, Portugal; Department of Chemistry and Biochemistry, University of Lisboa, Faculty of Sciences, Lisboa, United Kingdom, 5 Centro de Genetica Humana, Instituto Nacional Saude Dr Ricardo Jorge, Lisboa, Portugal

* To whom correspondence should be addressed. E-mail: H.Davidson{at}ed.ac.uk.

A panel of eleven human cystic fibrosis transmembrane conductance regulator (hCFTR) antibodies were tested in ovine nasal, tracheal and bronchial epithelial brushings. Two of these, G449 (polyclonal) and MATG1104 (monoclonal), recognised hCFTR but did not cross react with endogenous sheep CFTR. This specificity allows immunological detection of hCFTR expressed in gene transfer studies in sheep against the background of endogenous ovine CFTR, thus enhancing the value of the sheep as a model animal in which to study CFTR gene transfer. Studies on mixed populations of human and sheep nasal epithelial cells showed that detection of hCFTR by these two antibodies was possible even at the lowest proportion of human cells (1:100). The hCFTR gene was delivered in vivo by local instillation using polyethylenimine (PEI) -mediated gene transfer to the ventral surface of the ovine trachea and hCFTR mRNA and protein levels scored in a blinded fashion. Despite abundant hCFTR mRNA expression, the number of cells expressing hCFTR protein detectable by G449 was low (ca. 0.006-0.05%). Immunohistochemistry for hCFTR in animals treated by whole lung aerosol demonstrated positive cells in sections of tracheal epithelium and in distal conducting airways. The strategic employment of hCFTR specific antibodies supports the utility of the normal sheep as a model for hCFTR gene transfer studies.






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Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
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