An immunocytochemical assay to detect human CFTR expression following gene transfer.
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 w...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
2009
|
| _version_ | 1797052451153510400 |
|---|---|
| author | Davidson, H Wilson, A Gray, R Horsley, A Pringle, I McLachlan, G Nairn, A Stearns, C Gibson, J Holder, E Jones, L Doherty, A Coles, R Sumner-Jones, S Wasowicz, M Manvell, M Griesenbach, U Hyde, S Gill, DR Davies, J Collie, D Alton, E Porteous, D Boyd, A |
| author_facet | Davidson, H Wilson, A Gray, R Horsley, A Pringle, I McLachlan, G Nairn, A Stearns, C Gibson, J Holder, E Jones, L Doherty, A Coles, R Sumner-Jones, S Wasowicz, M Manvell, M Griesenbach, U Hyde, S Gill, DR Davies, J Collie, D Alton, E Porteous, D Boyd, A |
| author_sort | Davidson, H |
| collection | OXFORD |
| description | 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. CONCLUSION: 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. |
| first_indexed | 2024-03-06T18:31:45Z |
| format | Journal article |
| id | oxford-uuid:09e25980-c034-4ff9-8f96-841ff1e8965f |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:31:45Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | oxford-uuid:09e25980-c034-4ff9-8f96-841ff1e8965f2022-03-26T09:20:48ZAn immunocytochemical assay to detect human CFTR expression following gene transfer.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:09e25980-c034-4ff9-8f96-841ff1e8965fEnglishSymplectic Elements at Oxford2009Davidson, HWilson, AGray, RHorsley, APringle, IMcLachlan, GNairn, AStearns, CGibson, JHolder, EJones, LDoherty, AColes, RSumner-Jones, SWasowicz, MManvell, MGriesenbach, UHyde, SGill, DRDavies, JCollie, DAlton, EPorteous, DBoyd, A 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. CONCLUSION: 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. |
| spellingShingle | Davidson, H Wilson, A Gray, R Horsley, A Pringle, I McLachlan, G Nairn, A Stearns, C Gibson, J Holder, E Jones, L Doherty, A Coles, R Sumner-Jones, S Wasowicz, M Manvell, M Griesenbach, U Hyde, S Gill, DR Davies, J Collie, D Alton, E Porteous, D Boyd, A An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title | An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title_full | An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title_fullStr | An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title_full_unstemmed | An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title_short | An immunocytochemical assay to detect human CFTR expression following gene transfer. |
| title_sort | immunocytochemical assay to detect human cftr expression following gene transfer |
| work_keys_str_mv | AT davidsonh animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT wilsona animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT grayr animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT horsleya animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT pringlei animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT mclachlang animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT nairna animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT stearnsc animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT gibsonj animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT holdere animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT jonesl animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT dohertya animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT colesr animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT sumnerjoness animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT wasowiczm animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT manvellm animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT griesenbachu animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT hydes animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT gilldr animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT daviesj animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT collied animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT altone animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT porteousd animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT boyda animmunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT davidsonh immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT wilsona immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT grayr immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT horsleya immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT pringlei immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT mclachlang immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT nairna immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT stearnsc immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT gibsonj immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT holdere immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT jonesl immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT dohertya immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT colesr immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT sumnerjoness immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT wasowiczm immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT manvellm immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT griesenbachu immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT hydes immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT gilldr immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT daviesj immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT collied immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT altone immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT porteousd immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer AT boyda immunocytochemicalassaytodetecthumancftrexpressionfollowinggenetransfer |