Research Information
The Gene Expression Core was founded in 1994 and promotes translational, multidisciplinary CF research by furnishing expertise concerning recombinant CFTR and other proteins relevant to CF disease mechanism. Technically demanding protocols and reagents for cloning and propagating adenoviral and other recombinant viral systems, advances in gene suppression technology with sh- or siRNA, and capabilities for detection of CFTR mRNA and protein expression are also made available through the Core. These activities are organized through three Core functions:
Core Function #1: Clone new CFTR constructs and other CFTR regulatory proteins
The Core has the capabilities and skill to clone new CFTR constructs, including both common CFTR mutations and those that affect rare CFTR alleles that may be subject to unique cellular processing mechanisms. In addition, the Core can make constructs to express ENaC, TMEM16, or other proteins known to influence CFTR function. The Core maintains a repository of over 50 constructs containing CFTR and other relevant mutations, together with gene editing and other molecular tools useful to the CF community. These include CFTR-GFP fusions, HRP-fusions with extracellular CFTR loops, expression plasmids for individual CFTR domains (R-domain, nucleotide binding domains, portions of NBDs, carboxy and amino termini, second-site suppression alleles, cytoplasmic loops), eukaryotic expression plasmids (for synthesizing and purifying full length CFTR in mammalian cells), glycosylation mutants, epitope tags (HA, myc, FLAG), and over 50 clinical mutations or small deletions that cause human CF and are listed here.
Core Function #2: Create cell lines for investsigating CFTR biology and therapy
The Core generates and rigorously characterizes stable recombinant cell lines (created using both viral and plasmid-based protocols) expressing Core-cloned constructs for distribution throughout the CF community. These cell lines, provided across widely used durable cell types (e.g., FRT, Calu-3) and airway-specific cell expression systems emerging in their importance (e.g., 16HBE14o-, CFBE41o-), are essential for therapeutic drug screening targeting specific CFTR mutations as well as for studies of CF disease regulation. Currently available cell lines are listed here.
Core Function #3: Assist with studies of precise detection of CFTR mRNA and protein expression
Emerging technologies are improving our ability to quantify and characterize CFTR expression, including in primary cell, tissues, or diseased conditions that can be technically challenging and requisite of experience and expertise. To assist, the Core:
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Performs traditional methods such as PCR and Western blotting to measure CFTR expression
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Offers novel approaches to CFTR mRNA detection, including digital PCR and emerging RNAscope to localize nidus of expression necessary to understand CF pathogenesis and CFTR rescue. Ribosomal profiling is also offered.
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Maintains sh/siRNA and antisense oligonucleotide capabilities for knock-down and gene editing of CFTR and other cellular targets relevant to CF pathogenesis
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Provides a bridge to collaboration with single-cell RNA sequencing and analysis on campus
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Offers consultation for studies of CFTR biogenesis in experiments such as immunoblotting, immunoprecipitation, pulse-chase, cell surface biotinylation, and other biochemical means
Contact Information
Core users are associated both within and beyond the CF Research Center; there is no limitation on who may access the Core. For additional information regarding resources and services, please contact:
