Zoom Link
https://muw.zoom.us/j/96550703062
Department
Sciences & Mathematics
Format of Presentation
Oral Presentation
Research Category
STEM
Description
Cystic fibrosis (CF), primarily affecting Caucasians of northern European descent, results from mutations in the CFTR gene. DF508, the most common of CFTR mutations, results in dysfunctional Cl- channels in epithelial cells. Detecting CFTR protein expression at endogenous levels poses challenges, prompting this study to employ biochemical and molecular methods for its detection in non-transfected epithelial cell lines. Human pancreatic epithelial cell lines expressing endogenous wild-type CFTR (Capan-1) and DF508-CFTR (CFPAC) were treated with 5mM sodium butyrate for 60h to upregulate the CFTR expression. Additionally, a human lung epithelial cell line stably transfected with wild-type CFTR gene (CFBE-wt) was employed as a positive control. CFTR protein expression levels were assessed using biochemical techniques such as immunoprecipitation and improved western blotting [Heda et al, BioTechniques, 68(6), 319-325, 2020] using anti-CFTR polyclonal antibody MD1314. Concurrently, CFTR mRNA expression levels were determined by RT-qPCR, serving as a complementary approach to biochemical detection methods. The results demonstrated the successful detection of endogenous CFTR expression through both biochemical and molecular methodologies, where western blotting was employed for the first time in such detection. RT- qPCR emerged as the most convenient method, allowing us to quantitate the detection of endogenous CFTR expression, albeit at the mRNA level. However, biochemical detection of CFTR protein expression levels posed challenges due to the need for substantial quantities of antibodies and/or protein samples. Nevertheless, these detection methods hold promise for CF diagnosis using patient specimens.
Recommended Citation
Wilkins, Ethan, "COMPREHENSIVE DETECTION OF ENDOGENOUS CFTR EXPRESSION IN EPITHELIAL CELL LINES: BIOCHEMICAL AND MOLECULAR APPROACHES" (2025). Undergraduate Research Conference. 3.
https://athenacommons.muw.edu/urc/2025/oral-presentations-ii/3
COMPREHENSIVE DETECTION OF ENDOGENOUS CFTR EXPRESSION IN EPITHELIAL CELL LINES: BIOCHEMICAL AND MOLECULAR APPROACHES
Cystic fibrosis (CF), primarily affecting Caucasians of northern European descent, results from mutations in the CFTR gene. DF508, the most common of CFTR mutations, results in dysfunctional Cl- channels in epithelial cells. Detecting CFTR protein expression at endogenous levels poses challenges, prompting this study to employ biochemical and molecular methods for its detection in non-transfected epithelial cell lines. Human pancreatic epithelial cell lines expressing endogenous wild-type CFTR (Capan-1) and DF508-CFTR (CFPAC) were treated with 5mM sodium butyrate for 60h to upregulate the CFTR expression. Additionally, a human lung epithelial cell line stably transfected with wild-type CFTR gene (CFBE-wt) was employed as a positive control. CFTR protein expression levels were assessed using biochemical techniques such as immunoprecipitation and improved western blotting [Heda et al, BioTechniques, 68(6), 319-325, 2020] using anti-CFTR polyclonal antibody MD1314. Concurrently, CFTR mRNA expression levels were determined by RT-qPCR, serving as a complementary approach to biochemical detection methods. The results demonstrated the successful detection of endogenous CFTR expression through both biochemical and molecular methodologies, where western blotting was employed for the first time in such detection. RT- qPCR emerged as the most convenient method, allowing us to quantitate the detection of endogenous CFTR expression, albeit at the mRNA level. However, biochemical detection of CFTR protein expression levels posed challenges due to the need for substantial quantities of antibodies and/or protein samples. Nevertheless, these detection methods hold promise for CF diagnosis using patient specimens.
https://athenacommons.muw.edu/urc/2025/oral-presentations-ii/3