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Updated: Aug 15, 2026
Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Published on: March 10, 2012
Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes
R D Krauss1, G Berta, T A Rado
1Gregory Fleming James Cystic Fibrosis Research Center, Department of Microbiology, University of Alabama, Birmingham 35294.
Abstract:
Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.
Insights
Cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in lymphocyte function, even at low expression levels. Blocking CFTR in normal cells mimics the "CF phenotype," confirming its functional importance in lymphocytes.
Area of Science:
- Molecular Biology
- Cell Physiology
- Immunology
Background:
- Cystic fibrosis transmembrane conductance regulator (CFTR) is typically associated with epithelial cells but has roles in other cell types.
- Previous research indicated CFTR's involvement in adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes during the cell cycle.
Purpose of the Study:
- To investigate the functional presence and significance of CFTR in lymphocytes.
- To determine if low-level CFTR expression in lymphocytes impacts cell function.
Main Methods:
- Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to measure CFTR mRNA levels in lymphocytes.
- Western blot and immunoprecipitation to detect CFTR protein.
- Antisense oligonucleotides to inhibit CFTR function and assess effects on chloride permeability.
- 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy for measuring Cl- permeability.
Main Results:
- CFTR mRNA is present in the B lymphoid cell line GM03299, but at levels significantly lower (at least 1,000 times less) than in T84 cells.
- CFTR protein was undetectable by standard Western blot or immunoprecipitation methods.
- Antisense oligonucleotides targeting CFTR mRNA completely inhibited cell cycle-dependent Cl- permeability in normal lymphocytes, inducing a 'CF phenotype'.
Conclusions:
- A functional CFTR-associated Cl- permeability exists and is measurable in lymphocytes.
- Despite very low mRNA and undetectable protein levels, CFTR plays a measurable role in lymphocyte Cl- permeability.
- Inhibition of CFTR in normal lymphocytes replicates the functional defect seen in cystic fibrosis (CF) lymphocytes.
