Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines

Jefferson, Douglas M.; Valentich, J. D.; Marini, Frank C.; Grubman, Shelley A.; Iannuzzi, Michael C.; Dorkin, Henry L.; Li, Ming; Klinger, K.; Welsh, Michael J.

doi:10.1152/ajplung.1990.259.6.l496

Cited by 71 publications

(59 citation statements)

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“…JME cells were isolated from the nasal polyp of a CF patient with a homozyote ⌬508 genotype. 34 These cells retain the characteristics of the nasal epithelial cells and do not express any functional CFTR. 11,[34][35][36] The typical results of whole cell patch analyses of AAVp5-cftr transduced JME cells are shown in Figure 7.…”

Section: Correcting Cftr Defects In Cf Epithelial Cellsmentioning

confidence: 98%

“…34 These cells retain the characteristics of the nasal epithelial cells and do not express any functional CFTR. 11,[34][35][36] The typical results of whole cell patch analyses of AAVp5-cftr transduced JME cells are shown in Figure 7. A normal level of forskolinstimulated Cl currents (120 pA) were detected in cells transduced with AAVp5-cftr at MOI approximately 8.…”

Section: Correcting Cftr Defects In Cf Epithelial Cellsmentioning

confidence: 98%

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Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Wang

Fischer²,

Zhang

et al. 1999

Gene Ther

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Gene therapy studies of cystic fibrosis (CF) have shown ing capacity. Functional analyses showed that the new that AAV-based vector was efficient in transferring but not vectors were packaged efficiently and expressed higher in expressing the CFTR cDNA in the target cells. The levlevels of CFTR than a vector in which the CFTR gene was els of CFTR gene expression were limited by the small driven by the ITR sequence of AAV. Transduction of airway packaging capacity of AAV because it had been difficult to epithelial cells containing ٌ508 mutation with the new vecpackage the CFTR cDNA with an efficient promoter. In the tors demonstrated efficient expression of the wild-type present study we have developed a new generation of CFTR and correction of the CF phenotype. In contrast, no AAV/CFTR vectors which contain efficient short promoters significant CFTR expression was detected in cells infected to express the CFTR gene in target cells. To do so, we with the vector that express the CFTR gene from the ITR. reduced the size of the CFTR cDNA by determining the These findings support the notion that the AAV can be minimal untranslated regions required for expression of developed into an efficient vector to transduce the CFTR CFTR cDNA. We also identified short and efficient progene and vectors expressing higher levels of CFTR from moters that could be packaged with the down-sized CFTR an efficient promoter should provide better efficacy for cDNA into a novel AAV vector that had a maximal packaggene therapy of cystic fibrosis.

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Section: Correcting Cftr Defects In Cf Epithelial Cellsmentioning

confidence: 98%

Section: Correcting Cftr Defects In Cf Epithelial Cellsmentioning

confidence: 98%

Efficient CFTR expression from AAV vectors packaged with promoters – the second generation

Wang

Fischer²,

Zhang

et al. 1999

Gene Ther

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“…JME/CF15 nasal epithelia cells homozygous for ΔF508 CFTR (termed "CF15" throughout the manuscript) were cultured as described previously (22,24,25). For measuring redox potential using digital imaging microscopy cells were seeded on cover slips, transfected (Effectene, Qiagen) with plasmids coding for each roGFP.…”

Section: Cell Cultures Transfection and Infection Proceduresmentioning

confidence: 99%

“…We utilized the newly described ratiometric, redoxsensitive GFP1 (roGFP1, 20; 21) to measure redox potentials. roGFP1 was genetically targeted to the cytosol, endoplasmic reticulum (ER), mitochondria and apical surface in the CF nasal epithelial cell line CF15 (22), a cell line that is homozygous for the deletion of phenyl alanine 508 (ΔF508) and exhibits amiloride-sensitive Na + transport but no forskolinactivated Cl − secretion (23; 24). Quantitative measurements of roGFP1 fluorescence were performed using ratio imaging microscopy on living cells including calibrations of the fluorescence signals in terms of redox potentials.…”

Section: Introductionmentioning

confidence: 99%

Organelle redox of CF and CFTR-corrected airway epithelia

Schwarzer¹,

Illek²,

Suh³

et al. 2007

Free Radical Biology and Medicine

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In cystic fibrosis reduced CFTR function may alter redox properties of airway epithelial cells. Redox-sensitive GFP (roGFP1) and imaging microscopy were used to measure redox potentials of cytosol, ER, mitochondria and cell surface of cystic fibrosis nasal epithelial cells and CFTRcorrected cells. We also measured glutathione and cysteine thiol redox states in cell lysates and apical fluids to provide coverage over a range of redox potentials and environments that might be affected by CFTR. As measured with roGFP1, redox potentials at the cell surface (~ -207 ±8 mV) and in the ER (~ -217 ±1 mV) and rates of regulation of the apical fluid and ER lumen following DTT treatment were similar for CF and CFTR-corrected cells. CF and CFTR-corrected cells had similar redox potentials in mitochondria (-344 ±9 mV) and cytosol (-322 ±7 mV). Oxidation of carboxy-dichlorodihydrofluoresceindiacetate and of apical Amplex Red occurred at equal rates in CF and CFTR-corrected cells. Glutathione and cysteine redox couples in cell lysates and apical fluid were equal in CF and CFTR-corrected cells. These quantitative estimates of organelle redox potentials combined with apical and cell measurements using small molecule couples confirmed there were no differences in redox properties of CF and CFTR-corrected cells.

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“…We have investigated the potential rescue and stability at the cell membrane of F508-CFTR by VIP treatment in the human nasal epithelial cells JME/CF15, derived from a F508 homozygous patient (Jefferson et al, 1990). Immunostaining experiments with specific antiwww.intechopen.com CFTR antibodies, followed by confocal microscopy confirmed intracellular localization of F508-CFTR under control conditions, at 37°C, whereas membrane localization was observed in cells cultured at 27°C for 48hrs (Rafferty et al, 2009).…”

Section: Rescue Of F508-cftr Maturation and Membrane Stabilitymentioning

confidence: 96%