Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• Cystic fibrosis (CF) is a common inherited disorder in Caucasians.
• The DeltaF508 mutation in the CF transmembrane conductance regulator (CFTR) gene is present in 70% of CF chromosomes.
• Restoring functional CFTR is a key therapeutic goal.

Purpose of the Study:

• To achieve direct modification of the DeltaF508 mutation in endogenous CFTR using small fragment homologous replacement (SFHR).
• To generate and characterize human airway epithelial cells with corrected CFTR alleles.

Main Methods:

• Transfection of CFBE41o(-) cells (homozygous for DeltaF508) with wild-type (WT) CFTR DNA fragments using liposome-DNA complexes.
• Subcloning of transfected cells by limiting dilution and isolation of individual colonies.
• Analysis of DNA by allele-specific and non-allele-specific PCR, and genomic DNA fingerprinting.
• Assessment of CFTR mRNA expression using RT-PCR and sequence analysis.

Main Results:

• Detection of the CFTR-WT allele in five cell colonies using sensitive allele-specific PCR, indicating successful gene modification.
• DNA fingerprinting confirmed the isogenic nature of the colonies derived from the parental cell line.
• RT-PCR analysis revealed expression of WT-CFTR mRNA in some modified cell colonies, with one colony showing only WT mRNA expression.
• Sensitivity assays indicated that corrected cells constituted 1-10% of the total cell population.

Conclusions:

• SFHR successfully modified the DeltaF508 allele in homozygous human airway epithelial cells.
• The modified cells express functional WT-CFTR mRNA.
• Further subcloning is required to isolate pure isogenic clonal populations for therapeutic development.