Pérez-Luz Sara, Abdulrazzak Hassan, Grillot-Courvalin Catherine, Huxley Clare
NHLI, Imperial College London, London SW7 2AZ, UK.
Genomics. 2007 Nov;90(5):610-9. doi: 10.1016/j.ygeno.2007.07.005. Epub 2007 Sep 5.
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.
甲型血友病由编码凝血因子 VIII（F8）的基因突变引起，是基因治疗的重要靶点。F8 基因包含 26 个外显子，分布在约 186 kb 的区域，尚未有使用完整基因组位点的研究。我们构建了一个携带所有 26 个外显子、内含子以及超过 40 kb 上游和 20 kb 下游 DNA 的 250-kb BAC。这个 F8 BAC 进一步用来自 Epstein-Barr 病毒的 oriP/EBNA-1 元件进行改造，该元件可在哺乳动物细胞中实现附加体维持，或者用α卫星 DNA 进行改造，α卫星 DNA 可在一些人类细胞系中形成人类人工染色体。将含有 oriP/EBNA-1 的版本脂质转染到小鼠 Hepa1-6 细胞中，导致跨越 F8 基因的 F8 mRNA 表达。携带α卫星 DNA 的大于 300-kb BAC 通过细菌递送成功递送至 293A 和 HT1080 细胞，导致 F8 mRNA 表达水平高于内源性水平。
