Functional genomics assays to study CFTR traffic and ENaC function.

Faculty of Sciences, BioFiG-Centre for Biodiversity and Functional and Integrative Genomics, University of Lisboa, Lisboa, Portugal.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2011; 742:249-64. DOI: 10.1007/978-1-61779-120-8_15
Source: PubMed


As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here. These include high-content screens to determine proteins affecting function and trafficking of CFTR and ENaC.

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Available from: Margarida D Amaral
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    • "Our data also showed that inhibition of the latter reduces ENaC activity, leading to normalization of both Na + and fluid hyperabsorption in CF airways to non-CF levels in primary human lung cells from CF patients, supporting the potential role of this Na + channel as a therapeutic target. Currently, double-tagged wt-and F508del-CFTR constructs are being used [22] [23] "

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    • "Recently, Almaca and coworkers have published a fluorescent microscopy quantitative method that uses a CFTR chimera construct carrying mCherry plus a Flag epitope tag in CFTR 4th extracellular loop. In non-permeabilized cells, the Flag tag allows to quantify CFTR localized at the cell surface, while mCherry fluorescence indicates CFTR whole cell expression [19]. While this method provides a good dynamic range, it is limited to proteins with extracellular domains. "
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    • "Automated Microscopy and High-Content siRNA Screening Cells ($150,000 cells) were plated in 1.5 ml of culture medium in siRNA prespotted chambered slides. After 48 hr, cells were incubated for 10 min with FMP as described (Almaç a et al., 2011). Cells on siRNA spots (1-384) were imaged with 103 objective in Cy3 channel, the FMP solution was then automatically replaced by FMP/Amil; after 3 min, the same image acquisition routine started. "
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    ABSTRACT: Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na(+) and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
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