1. Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination.
- Author
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Pérez-Luz S, Abdulrazzak H, Grillot-Courvalin C, and Huxley C
- Subjects
- Animals, Carcinoma, Hepatocellular genetics, Carcinoma, Hepatocellular metabolism, Carcinoma, Hepatocellular pathology, Cells, Cultured, DNA analysis, Factor VIII metabolism, Fibrosarcoma genetics, Fibrosarcoma metabolism, Fibrosarcoma pathology, Gene Targeting, Genetic Vectors, Humans, Kidney metabolism, Liver Neoplasms genetics, Liver Neoplasms metabolism, Liver Neoplasms pathology, Mice, Plasmids, Polymerase Chain Reaction, RNA, Messenger metabolism, Transfection, Chromosomes, Artificial, Bacterial, DNA genetics, Epstein-Barr Virus Nuclear Antigens genetics, Factor VIII genetics, RNA, Messenger genetics, Recombination, Genetic
- Abstract
Hemophilia A is caused by mutations in the gene encoding factor VIII (F8) and is an important target for gene therapy. The F8 gene contains 26 exons spread over approximately 186 kb and no work using the intact genomic locus has been carried out. We have constructed a 250-kb BAC carrying all 26 exons, the introns, and more than 40 kb of upstream and 20 kb of downstream DNA. This F8 BAC was further retrofitted with either the oriP/EBNA-1 elements from Epstein-Barr virus, which allow episomal maintenance in mammalian cells, or alphoid DNA, which allows human artificial chromosome formation in some human cell lines. Lipofection of the oriP/EBNA-1-containing version into mouse Hepa1-6 cells resulted in expression of F8 mRNA spanning the F8 gene. The >300-kb BAC carrying alphoid DNA was successfully delivered to 293A and HT1080 cells using bacterial delivery, resulting in greater than endogenous levels of F8 mRNA expression.
- Published
- 2007
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