Research Information
The CF Center’s Gene Editing and Expression Core (Core B) was founded in 1994 and promotes translational, multidisciplinary CF research by furnishing expertise concerning recombinant CFTR and other proteins relevant to CF disease mechanism. Core B maintains a strong focus on both translational and basic science aspects of CF molecular and cellular biology on behalf of our extensive CF research community. The Core serves as a key bridge connecting basic science to translational studies by providing essential constructs and cell lines to CF investigators. Using cutting-edge gene cloning and editing tools, Core B: 1) creates constructs expressing CFTR containing both common CFTR mutations and more rare mutations that affect the remaining 7-10% of CFTR to study their responses to CFTR modulators and other developing therapeutic reagents; 2) generates cell lines expressing CFTR-interacting proteins that may affect CFTR mRNA expression and function; 3) creates more advanced gene-editing reagents for CFTR gene correction; 4) makes rigorously characterized recombinant cell lines which have been instrumental for ongoing drug discovery efforts at UAB and elsewhere; and 5) examines the patterns of CFTR expression and biogenesis in airway and other epithelial cell types. Each of these capabilities is essential to maintain major research themes in our Center and augment research areas within the mission of the RDP. Since emerging methods for gene editing and delivery are often technically challenging and time-consuming for individual laboratories, Core B conducts such protocols and provides trainings for both experienced and new CF scientists. This tight coordination builds a community of multidisciplinary CF research faculty. The objectives of Core B are as follows:
Core Function #1: Generate constructs and cell lines expressing CFTR variants and reporters to facilitate the drug discovery for the treatment of CFTR rare mutants
~5% patients with CF (PwCF) carry >90 premature stop codon (PTC) mutations that are not responsive to CFTR modulators. Small molecule readthrough (RT) drugs have been actively pursued to treat these PwCF. To help the development of the RT-drugs and understand the response of different CFTR PTCs to various treatment, Core B made reporter constructs containing different CFTR PTCs in their native genetic context. Core B also made cell lines expressing CFTR variants that have near cognate amino acids inserted to PTCs to test their channel function. In addition, the expression of CFTR hinges on cis elements surrounding the CFTR gene locus. Core B is responsible for making novel CFTR constructs with specific introns and 3’-UTR regions to study their effect on regulating CFTR gene expression. Furthermore, CFTR function is known to be affected by other proteins such as nonsense mediated decay (NMD) factors, the unfolded protein response, and even other ion channels. We have generated, characterized, and distributed stable cell lines to study the function of these cis- and trans- factors on CFTR gene expression. These cell lines are essential for therapeutic drug discovery that targets specific CFTR mutations, which is critical for studies of CF disease regulation and is thematically important to our Center’s mission. Core B will continuously provide cell lines (e.g. FRT, 16HBE14o- and CFBE41o-) expressing other exceedingly rare CFTR mutants.
Core Function #2: Provide novel gene-editing tools and protocols to facilitate the studies using primary airway epithelium, basal cells and iPS cells.
The Core B has been adding new highly effective gene-editing techniques to modify CFTR and CFTR associated genes in primary airway epithelium, basal cells, and iPS cells in our CF Research Center. These cells enable investigators to better understand the consequences of modulator treatments, read-through agents, and genetic based therapies as well as to study CF biology and pathology in cell culture models closer to the in vivo physiological condition. Lentiviral and AAV-based vectors have been developed for both CRISPR/Cas9-mediated editing and prime-editing to achieve highly effective gene modification. In collaboration with Core D, we are generating basal cells and iPS cells carrying specific CFTR mutations (e.g. PTCs), knock-out or introduce mutations in CFTR modifier genes, or knock-in reporters into endogenous genes.
Core Function #3: Provide cellular and molecular analysis tools to monitor gene-editing and other CF therapies.
Emerging technologies are improving our ability to quantify and characterize CFTR expression in primary cells and tissues. However, these techniques can be technically challenging, requiring specialized experience and expertise to perform. In addition to traditional methods such as PCR and Western blotting to measure CFTR expression, Core B provides novel capability of detecting CFTR mRNA abundance more precisely using digital PCR and RNAscope technology to better understand CFTR expression in studies of CF pathogenesis and CFTR rescue. Core B also maintains sh/siRNA, CRISPR/Cas9 and antisense oligonucleotide capabilities for performing knock-down experiments, and gene editing of CFTR and other cellular targets relevant to CF pathogenesis. We will also help with indel and off-target analysis after gene-editing of different cell lines. Finally, the Core provides daily consultations for studies of CFTR biogenesis by providing techniques such as immunoblotting, immunoprecipitation, pulse-chase, cell surface biotinylation, ribosome profiling and other biochemical approaches essential for rigorous CF research.
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:
