Cystic Fibrosis (CF) is a genetic disease featuring a mutation in the CFTR gene that leads to a buildup of thick clumps of high-molecular-weight glycoproteins (mucins/MUCs) in the lungs and the intestines. This causes difficulty breathing, frequent infections, poor growth, nutrient malabsorption, and ultimately death. Although pulmonary complications are the most life threatening and the most studied, investigating intestinal physiology may provide better insight into the altered mucus properties and systemic effects of CFTR dysfunction. Our goal was to establish a model system to study the effects of CFTR function on gut mucus and to further characterize mucus properties in the intestine. The HT29 cell line, derived from a colon adenocarcinoma, was used in this project as it has previously proven to express both CFTR and mucin genes. To determine the most appropriate culture conditions and optimize expression of mucins and CFTR, HT29 cells were grown in different media, and the validity of the model was evaluated through resistance measurements, mucin biochemical assays (e.g., immunohistochemical staining and Western blotting), and histology. CFTR Cl- secretion was measured by Ussing chamber following forskolin stimulation, but the formation of an intercellular lumina “blister” impeded the traditional measurement of CFTR function. Following culture conditions optimization, a CFTR knockout was generated through CRISPR/Cas9 lentivirus infection and confirmed via protein analysis. In parallel, healthy wild-type (WT) cells were compared to cells where CFTR was pharmacologically inhibited (i.e., CF-like cells) and showed differences in mucin concentrations. Our data suggests these mucin-producing cell lines can function as relevant in vitro models to study the CF gut mucus phenotype. Investigating pure mucus in an environment devoid of inflammatory cells and/or fecal matter can help focus on mucins’ abnormal properties and reveal novel pharmaceutical targets. A treatment targeting mucins or reversing the abnormal mucus phenotype would be mutation agnostic (i.e., independent of genotype) and therefore would have the potential to slow the progression of the disease and benefit all patients with CF.Bachelor of Scienc
