CFTR expression from a BAC carrying the complete human gene and associated regulatory elements.

Area of Science:

• Genetics
• Molecular Biology
• Gene Therapy

Background:

• Genomic DNA offers advantages for gene therapy due to regulatory elements.
• No bacterial artificial chromosome (BAC) containing the entire cystic fibrosis transmembrane conductance regulator (CFTR) gene was previously available.

Purpose of the Study:

• To construct a BAC vector containing the complete human CFTR gene for gene therapy applications.
• To evaluate the expression and functionality of the CFTR gene within the BAC construct.

Main Methods:

• Red homologous recombination was used to create a BAC vector with a 250.3-kb insert of the CFTR gene, including flanking regulatory regions.
• RT-PCR and sequencing were employed to assess gene expression and mRNA splicing in CMT-93 cells.

Main Results:

• The constructed BAC vector successfully expressed the CFTR gene in CMT-93 cells, both from integrated copies and episomes.
• Sequencing confirmed that the BAC can generate correctly spliced human CFTR mRNA in a mouse cell background.
• This BAC represents the only available genomic CFTR construct on a convenient vector.

Conclusions:

• The developed BAC vector is a functional genomic construct for the CFTR gene.
• This tool is suitable for gene expression studies and in vivo testing for cystic fibrosis gene therapy.