Transgenic complementation studies on cystic fibrosis mouse

Oceandy, Delvac. (2002). Transgenic complementation studies on cystic fibrosis mouse PhD Thesis, School of Molecular and Microbial Sciences, The University of Queensland.

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Author Oceandy, Delvac.
Thesis Title Transgenic complementation studies on cystic fibrosis mouse
School, Centre or Institute School of Molecular and Microbial Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Brandon Wainwright
David Hume
Total pages 148
Language eng
Subjects 06 Biological Sciences
Formatted abstract
Lung disease is the leading cause of morbidity in cystic fibrosis (CF). Chronic endobronchial infection by Pseudomonas aeruginosa and excessive inflammatory response are the main features of CF lung disease. Defective cystic fibrosis transmembrane conductance regulator (CFTR) function in airway epithelial cells contribute directly to the retention of P.aeruginosa infection in CF lung. CF epithelial cells and macrophages also showed abnormal inflammatory response after treatment with P.aeruginosa compared to normal cells. Therefore, correction of CFTR activity may be required in those cells to fully ameliorate such abnormalities.

Transgenic complementation studies were conducted to further elucidate the role of airway epithelial cells and alveolar macrophages in CF lung disease. In this study, I generated animal models of genetic reconstitution in which the human CFTR gene is expressed exclusively in lung epithelia or alveolar macrophages of G551D CF mice. The G551D CF mouse model has CF characteristic phenotypes including altered chloride conduction in airways and intestine, dysregulated lung inflammatory response and altered bacterial clearance in the lung. The epithelial specific promoter keratin 18 (K18) and macrophage specific promoter A6.7fnis were used to direct the expression of human CFTR in the G551D mice. The bitransgenic mice are referred to as G551D-K18 for the G551D mice expressing human CFTR in airway epithelial cells, and G55IDfins for the G551D mice expressing human CFTR in macrophages.

To model chronic endobronchial infection, P.aeruginosa were entrapped in agar beads and instilled into the lung of bitiansgenic G551D-K18, G551D-fms and also to normal mice and G551D mice. Bacterial survival in the lung was determined and the concentration of proinflammatory cytokines in the bronchoalveolar lavage fluid was also evaluated at 3 days postinfection. I found that transgenic expression of the human CFTR gene in airway epithelia by using KI8 promoter corrected the bacterial clearance defect in G551D CF mice. In contrast, expression of the same transgene in macrophages using the A6.7fms promoter did not correct the defect. Moreover, the levels of inflammatory mediators in bronchoalveolar lavage fluid was reduced to normal levels in the G551D-K18 mice.

To test whether the defect in CFTR is related directly to the development of excessive inflammation and whether it can be corrected by transgenic complementation, the mouse models were stimulated with Pseudomonas lipopolysaccharide (LPS) so that the inflammatory stimuli is independent from the bacterial clearance capacity. The LPS were injected intratracheally and after 6 hours several inflammatory indices were examined in the lung. I found that the expression of S100A8, which is a surrogate marker of neutrophil infiltration, was elevated in G551D mice. In the G551D-K18 mice, the S100A8 expression was significantly reduced. However, the G55 ID-fins still exhibited high S100A8 expression.

These results suggest that replacement of CFTR in the airway epithelial cells can lead to the improvement of bacterial clearance and inflammatory response in the lung of 055ID CF mice. Further, these findings indicate for the first time which cell types need to be corrected to achieve therapeutic benefits and demonstrate the potential of gene replacement approach in developing new therapy for CF lung disease.
Keyword Cystic fibrosis.

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
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Created: Tue, 01 Mar 2011, 11:37:14 EST by Ning Jing on behalf of Social Sciences and Humanities Library Service