Expression of the human CFTR gene from episomal oriP-EBNA1-YACs in mouse cells

• Published in: Human molecular genetics 2000-03, Vol.9 (4), p.617-629
• Main Authors: HUERTAS, D, HOWE, S, MCGUIGAN, A, HUXLEY, C
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Animals; Biological and medical sciences; Cell Line; CFTR gene; Chromosomes - metabolism; Chromosomes, Artificial, Yeast - genetics; Chromosomes, Artificial, Yeast - metabolism; Cystic Fibrosis Transmembrane Conductance Regulator - biosynthesis; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; EBNA-1 gene; EBNA-1 protein; Epstein-Barr virus; Epstein-Barr Virus Nuclear Antigens - biosynthesis; Epstein-Barr Virus Nuclear Antigens - genetics; Epstein-Barr Virus Nuclear Antigens - metabolism; Gastroenterology. Liver. Pancreas. Abdomen; Gene Dosage; Gene Expression Regulation; Glycine - genetics; Humans; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Medical sciences; Mice; Other diseases. Semiology; Plasmids - chemical synthesis; Plasmids - genetics; Plasmids - metabolism; Recombination, Genetic; Repetitive Sequences, Amino Acid; Replication Origin - genetics; Transgenes; transmembrane conductance regulator; Tumor Cells, Cultured; yeast artificial chromosomes
• ISSN: 0964-6906; 1460-2083; 1460-2083
• DOI: 10.1093/hmg/9.4.617

Plasmids carrying the origin of plasmid replication ( oriP ) and expressing the EBNA-1 protein from the Epstein-Barr virus replicate and segregate in human cells and are thus potentially useful vectors for gene therapy. As very large circular molecules, up to 660 kb in size, can be maintained episomally using this system, it is possible to include intact human genes with all their long-range controlling elements which might give high levels of tissue-specific and controlled gene expression. We have shown previously that a 320 kb yeast artificial chromosome (YAC) carrying the intact human CFTR gene can complement the Cambridge null cystic fibrosis mice as a transgene. We have now modified this YAC to a circular molecule carrying both oriP and the EBNA-1 gene. We show that this oriP-EBNA1-YAC can be stably maintained as unrearranged episomes in mouse LA-9 cells, which do not express endogenous cftr, and in mouse CMT-93 cells, which do express endogenous cftr. The human CFTR gene is expressed in some of the cell lines, but the level of expression is very variable between cell lines and is not related to the copy number of the elements.