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...
| Asıl Yazarlar: | , , , , , , , , , , , , , , , , |
|---|---|
| Materyal Türü: | Journal article |
| Dil: | English |
| Baskı/Yayın Bilgisi: |
2010
|
| _version_ | 1826259505028005888 |
|---|---|
| author | Holder, E Stevenson, B Farley, R Hilliard, T Wodehouse, T Somerton, L Larsen, M O'Donoghue, J Coles, R Scheule, R Cheng, S Gill, DR Hyde, S Griesenbach, U Alton, E Porteous, D Boyd, A |
| author_facet | Holder, E Stevenson, B Farley, R Hilliard, T Wodehouse, T Somerton, L Larsen, M O'Donoghue, J Coles, R Scheule, R Cheng, S Gill, DR Hyde, S Griesenbach, U Alton, E Porteous, D Boyd, A |
| author_sort | Holder, E |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T18:50:57Z |
| format | Journal article |
| id | oxford-uuid:1030af71-8f16-47fb-8049-bb34b65dbf3e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:50:57Z |
| publishDate | 2010 |
| record_format | dspace |
| spelling | oxford-uuid:1030af71-8f16-47fb-8049-bb34b65dbf3e2022-03-26T09:55:12ZDetection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:1030af71-8f16-47fb-8049-bb34b65dbf3eEnglishSymplectic Elements at Oxford2010Holder, EStevenson, BFarley, RHilliard, TWodehouse, TSomerton, LLarsen, MO'Donoghue, JColes, RScheule, RCheng, SGill, DRHyde, SGriesenbach, UAlton, EPorteous, DBoyd, ABACKGROUND: 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. |
| spellingShingle | Holder, E Stevenson, B Farley, R Hilliard, T Wodehouse, T Somerton, L Larsen, M O'Donoghue, J Coles, R Scheule, R Cheng, S Gill, DR Hyde, S Griesenbach, U Alton, E Porteous, D Boyd, A Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title | Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title_full | Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title_fullStr | Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title_full_unstemmed | Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title_short | Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity. |
| title_sort | detection of cftr transgene mrna expression in respiratory epithelium isolated from the murine nasal cavity |
| work_keys_str_mv | AT holdere detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT stevensonb detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT farleyr detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT hilliardt detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT wodehouset detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT somertonl detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT larsenm detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT odonoghuej detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT colesr detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT scheuler detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT chengs detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT gilldr detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT hydes detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT griesenbachu detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT altone detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT porteousd detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity AT boyda detectionofcftrtransgenemrnaexpressioninrespiratoryepitheliumisolatedfromthemurinenasalcavity |