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Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination. 2007

Sara Pérez-Luz, and Hassan Abdulrazzak, and Catherine Grillot-Courvalin, and Clare Huxley
NHLI, Imperial College London, London SW7 2AZ, UK. saraperezluz@yahoo.co.uk

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.

UI MeSH Term Description Entries
D007668 Kidney Body organ that filters blood for the secretion of URINE and that regulates ion concentrations. Kidneys
D008113 Liver Neoplasms Tumors or cancer of the LIVER. Cancer of Liver,Hepatic Cancer,Liver Cancer,Cancer of the Liver,Cancer, Hepatocellular,Hepatic Neoplasms,Hepatocellular Cancer,Neoplasms, Hepatic,Neoplasms, Liver,Cancer, Hepatic,Cancer, Liver,Cancers, Hepatic,Cancers, Hepatocellular,Cancers, Liver,Hepatic Cancers,Hepatic Neoplasm,Hepatocellular Cancers,Liver Cancers,Liver Neoplasm,Neoplasm, Hepatic,Neoplasm, Liver
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D011995 Recombination, Genetic Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses. Genetic Recombination,Recombination,Genetic Recombinations,Recombinations,Recombinations, Genetic
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D005169 Factor VIII Factor VIII of blood coagulation. Antihemophilic factor that is part of the factor VIII/von Willebrand factor complex. Factor VIII is produced in the liver and acts in the intrinsic pathway of blood coagulation. It serves as a cofactor in factor X activation and this action is markedly enhanced by small amounts of thrombin. Coagulation Factor VIII,Factor VIII Clotting Antigen,Factor VIII Coagulant Antigen,Factor VIII Procoagulant Activity,Thromboplastinogen,Blood Coagulation Factor VIII,F VIII-C,Factor 8,Factor 8 C,Factor Eight,Factor VIIIC,Hyate-C,Hyatt-C,F VIII C,Hyate C,HyateC,Hyatt C,HyattC
D005354 Fibrosarcoma A sarcoma derived from deep fibrous tissue, characterized by bundles of immature proliferating fibroblasts with variable collagen formation, which tends to invade locally and metastasize by the bloodstream. (Stedman, 25th ed) Fibrosarcomas
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D006528 Carcinoma, Hepatocellular A primary malignant neoplasm of epithelial liver cells. It ranges from a well-differentiated tumor with EPITHELIAL CELLS indistinguishable from normal HEPATOCYTES to a poorly differentiated neoplasm. The cells may be uniform or markedly pleomorphic, or form GIANT CELLS. Several classification schemes have been suggested. Hepatocellular Carcinoma,Hepatoma,Liver Cancer, Adult,Liver Cell Carcinoma,Liver Cell Carcinoma, Adult,Adult Liver Cancer,Adult Liver Cancers,Cancer, Adult Liver,Cancers, Adult Liver,Carcinoma, Liver Cell,Carcinomas, Hepatocellular,Carcinomas, Liver Cell,Cell Carcinoma, Liver,Cell Carcinomas, Liver,Hepatocellular Carcinomas,Hepatomas,Liver Cancers, Adult,Liver Cell Carcinomas

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