Escherichia coli-cloned CFTR loci relevant for human artificial chromosome therapy.

Classical gene therapy for cystic fibrosis has had limited success because of immune response against viral vectors and short-term expression of cDNA-based transgenes. These limitations could be overcome by delivering the complete genomic CFTR gene on nonintegrating human artificial chromosomes (HACs). Here, we report reconstruction of the genomic CFTR locus and analyze incorporation into HACs of three P1 phage-based and F factor bacteria-based artificial chromosomes (PACs/BACs) of various sizes: (1) 5A, a large, nonselectable BAC containing the entire wild-type CFTR locus extending into both adjacent genes (296.

Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei.

We investigated in different human cell types nuclear positioning and transcriptional regulation of the functionally unrelated genes GASZ, CFTR, and CORTBP2, mapping to adjacent loci on human chromosome 7q31. When inactive, GASZ, CFTR, and CORTBP2 preferentially associated with the nuclear periphery and with perinuclear heterochromatin, whereas in their actively transcribed states the gene loci preferentially associated with euchromatin in the nuclear interior. Adjacent genes associated simultaneously with these distinct chromatin fractions localizing at different nuclear regions, in accordance with their individual transcriptional regulation.