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Updated: Jul 11, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination
Sara Pérez-Luz1, Hassan Abdulrazzak, Catherine Grillot-Courvalin
1NHLI, Imperial College London, London SW7 2AZ, UK. saraperezluz@yahoo.co.uk
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.
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