Introduction of human mini-chromosome vector carrying human dystrophin gene into the mdx mice

Duchenne肌营养不良症(DMD)是一种严重的X连锁隐性进行性肌肉萎缩疾病，由DMD基因突变引起，该基因编码一种名为dystrophin的427 kDa细胞骨架蛋白。抗肌营养不良蛋白基因非常复杂；79个外显子分布在大约2.5mb的基因组区域，至少有7个单独的启动子。在本实验中，我们试图将人类X染色体上的整个dystrophin基因组区域导入到mdx小鼠中，这是一种X染色体连锁的隐性肌病突变体，用作人类DMD模型。首先，我们从mdx小鼠中分离出胚胎干细胞，建立了一株新的ES细胞系，命名为mdx-1。MDX-1 ES细胞具有形成有活力的生殖系嵌合体的能力。接下来，利用Cre-loxP系统，将人类X染色体的整个Dystrophin基因组区域转移到稳定的人类微染色体载体HAC-SC20中。然后通过微细胞介导的染色体转移技术将含dystrophin的微染色体导入MDX-1 ES细胞。通过将ES细胞注射到MDX囊胚中，我们产生了嵌合小鼠，在其中人类抗肌营养不良蛋白基因可能是唯一表达的。

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive, progressive muscle-wasting disease caused by mutations in the DMD gene that encodes a 427-kDa cytoskeletal protein called dystrophin. The dystrophin gene is uniquely complex ; 79 exons spread, over approximately 2.5Mb of genomic region, with at least seven separate promoters. Multiple isoforms of dystrophin, thus formed, are expressed in muscle and non-muscle tissues at separate stages of development.In the present experiment, we have attempted to introduce whole dystrophin genomic region of the human X chromosome into the mdx mice, which is X chromosome-linked recessive myopathic mutants used as human models of DMD. At first, we have isolate embryonic stem cells from the mdx mice and established a new ES cell line, named mdx-1. The mdx-1 ES cells had the ability of forming viable germ line chimeras. Next, whole dystrophin genomic region of the human X chromosome was allowed to translocate, utilizing Cre-loxP system, into a stable human minichromosome vector, HAC-SC20. The dystrophin-cairying minichromosome was then transferred into the mdx-1 ES cells by microcell-mediated chromosome transfer technique. By injecting the ES cells into the mdx blastocysts, we have produced chimeric mice, in which human dystrophin gene might be expressing exclusively.