%0 Journal Article
%A Pérez Luz, Sara
%A Abdulrazzak, Hassan
%A Grillot-Courvalin, Catherine
%A Huxley, Clare
%T Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination
%D 2007
%@ 0888-7543
%U https://hdl.handle.net/20.500.14352/93001
%X 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 ∼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.
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