WS15.1 Development of a human model of airway epithelial cells invalidated for CFTR

2013 
Objectives: The sequence of events occurring at the early onset of pulmonary infection and inflammation is poorly understood. Primary cultures of well-differentiated CF human airway epithelial cells (HAECs) are valuable but difficult to generate and heterogeneous. Our objective is to genetically modify non-CF HAECs by disrupting the CFTR gene and create a CF-like 3D model of the airway epithelium. Methods: shRNA sequences targeting the CFTR mRNA (shCFTR) were screened and the three best silencing sequences were cloned as single and triple constructs to produce lentiviral (LV) vectors co-expressing the green fluorescent protein. Their efficiency was assessed by transduction of Calu-3 airway epithelial cells. GFP positive cells sorted by FACS were cultured on filters for polarization to measure transepithelial currents in Ussing chambers and CFTR protein levels by Western blot. Preliminary results showed a partial knock-down of the CFTR protein coupled with variations in the transepithelial currents. Primary HAECs were also transduced with LV vectors expressing GFP only or a shRNA sequence targeting the gap junction protein Connexin26. Immunofluorescence analyses showed a good knockdown efficiency of Cx26 while GFP remained expressed in cells grown at the air-liquid interface for at least 30 days. Conclusion: These results validate our strategy to knock-down CFTR sequences in primary HAECs. Experiments are ongoing to identify more efficient shCFTR sequences. Ultimately, we expect to investigate the phenotypic consequences of CFTR knock-down/knock-out compared to the parental cells, therefore eliminating the variability of CF specimen.
    • Correction
    • Source
    • Cite
    • Save
    • Machine Reading By IdeaReader
    0
    References
    0
    Citations
    NaN
    KQI
    []