Gene analysis and gene therapy in tyrosinemias.

我们分别从人、小鼠和猪的肝脏mRNA构建的cDNA文库中克隆了人、小鼠和猪的4-羟苯丙酮酸双加氧酶的cDNA，并以人和小鼠的cDNA为对照，从人和小鼠基因组文库中获得了含有HPD外显子的DNA片段，通过这些研究，阐明了人和小鼠HPD基因的结构。在此基础上，我们对日本1型酪氨酸血症患者的FAH基因和酪氨酸血症小鼠III型小鼠的HPD基因进行了基因突变分析，分别发现了FAH基因和HPD基因的突变。我们产生了携带两种突变的小鼠，一种在FAH基因上，一种在HPD基因上。Fah基因缺陷小鼠具有致死性，而这些双突变体却具有致死性，说明HPD基因缺陷挽救了白化病缺失小鼠的致死性表型。进一步构建了表达人HPD的重组腺病毒，在培养的细胞中转导，重组病毒可有效地表达人HPD，为在实验动物中表达人HPD奠定了基础。

We have clone cDNA for human, mouse and porcine 4-hydroxyphenylpyruvate dioxygenase from cDNA libralies constructed from mRNA of human, mouse and porcine liver respectively, Using human and mouse cDNA as proves, we obstained DNA fragments containing exons of HPD from human and mouse genomic libraries, Through these studies we have clarified the structure of human and mouse HPD gene. Similarly, we have elucidated the structures of cDNA and gene for human fumarylacetoacetase.Based on these progress we analyzed gene mutations in human FAH gene from a Japanese patient with type 1 tyrosinemia and mouse HPD gene from III mouse, a tyrosinemic mouse.These studies revealed mutations on FAH gene and HPD gene, respectively.Using abino lethal mouse which is defective for Fah, and III mouse which is defective for HPD,we generated mice which carry both mutation, one on FAH gene and one on HPD gene. Fah deficient mice were neonatally lethal but these double mutants were vaiable, indicating that defective HPD gene rescues the lethal phenotype of albino deletion mouse.Futher, we constructed recombinant adenovirus which express human HPD.In cultured cells, transduction of the recombinant virus effectively expresses human HPD.This recombinant virus will be useful to express human HPD in experimental animals.