ANIMAL MODEL OF HOMOCYSTINURIA BY GENE EXCISION

这项研究的长期目标是培育小鼠品系 用改变或缺失的胱硫醚β-合酶基因(S) 模型同型半胱氨酸尿症和早产儿外周及脑血管疾病 (PPAD/CeVD)。哥伦比亚广播公司是一种核心的酶 含硫氨基酸的硫化/再甲基化支点 新陈代谢。它是一个由63 kDa亚基组成的四聚体。它的基因驻留在 人类21号染色体和小鼠17号染色体--遗传性CBS缺乏症 导致最常见的同型半胱氨酸尿症；这种情况是 在美国的一些州，现在被诊断的频率比北大更高 新生儿筛查计划。同型半胱氨酸尿症伴有精神症状 发育迟缓、癫痫大发作、视晶状体脱位、骨骼 精神紊乱和致命的血管疾病。约三分之一的未经选择的患者 PPAD/CEAD是CBS缺乏症的杂合子。那里 目前还没有针对这些情况的动物模型。的最新进展 胚胎干细胞中同源重组的基因切除 结合我们对CBS基因序列和组织的研究 将使我们能够培育出缺乏一种或两种CBS等位基因的动物。 我们的具体目标是：1)评估小鼠CBS基因以便于 靶向序列的分子克隆；2)构建这样的小鼠 CBS靶向序列将切除大约一半的鼠标 3)制备CBS等位基因缺失的ES细胞 与该序列进行同源重组，并利用这些细胞 产生杂合子和纯合子小鼠；4)病理生理学检查 和纯合子CBS缺乏症动物模型的生物化学；5) 确定半正常CBS基因剂量是否与 动物模型中的PPAD/CeVD；以及6)替换转录部分 将小鼠CBS基因与正常和突变的人CBS基因进行比较，并对 评估人类突变对动物的影响。蜂窝， 分子、生化、解剖学和发育方法将是 应用包括：i)通过同源重组切除基因 胚胎干细胞；ii)胚胎干细胞产生嵌合体小鼠 胚泡定植；III)杂合子和纯合子的选育 动物模型；iv)通过聚合酶链式反应、核酸技术评估CBS基因的表达 酸和蛋白质印迹以及酶分析；v)粗面和 目标组织的显微镜检查；vi)神经学、行为学 并对由此产生的动物进行发育分析。此外， 动物模型将提供给其他进行实验的实验室 同型半胱氨酸尿症与PPAD/CeVD的研究这些研究将阐明 硫磺氨基酸代谢紊乱；此外，他们将允许 CBS缺乏与慢性阻塞性肺疾病临床表现的相关性 并应促进生物化学原理的发展 用于治疗某些突变情况。

The long-term objective of this research is to develop strains of mice with altered or deleted cystathionine beta-synthase (CBS) gene(s) to model homocystinuria and premature peripheral and cerebrovascular disease (PPAD/CeVD). CBS is a central enzyme at the transsulfuration/remethylation branch point of sulfur amino acid metabolism. It is a tetramer of 63 kDa subunits. Its gene resides on human chromosome 21 and mouse chromosome 17. Inherited CBS deficiency precipitates the most common form of homocystinuria; this condition is now being diagnosed more frequently than PKU in some American states with neonatal screening programs. Homocystinuria is accompanied by mental retardation, grand mal seizures, dislocated optic lenses, skeletal disorders, and fatal vascular episodes. About 1/3 of unselected patients with PPAD/CeAD are reported to be heterozygous for CBS deficiency. There are currently no animal models for these conditions. Recent advances in gene excision via homologous recombination in embryonic stem cells together with our studies of the sequence and organization of CBS genes will allow us to develop animals deficient in one or both cbs alleles. Our specific aims are: 1) To evaluate the mouse CBS gene to facilitate molecular cloning of the targeting sequence; 2) to construct such a mouse CBS targeting sequence which will excise approximately half of the mouse CBS gene; 3) to prepare ES cells deficient in one CBS allele by homologous recombination with this sequence, and to use these cells to generate hetero- and homozygous mice; 4) to examine the pathophysiology and biochemistry of homozygous CBS deficiency in animal model; 5) to determine whether half-normal CBS gene dosage is associated with PPAD/CeVD in the animal model; and 6) to replace the transcribed portion of the mouse CBS gene with normal and mutant human CBS cDNA, and to evaluate the effects of human mutations on the animal. Cellular, molecular, biochemical, anatomical, and developmental approaches will be utilized including: i) Gene excision by homologous recombination in embryonic stem cells; ii) generation of chimeric mice by ES cell blastocyst colonization; iii) breeding of the heterozygous and homozygous animal models; iv) evaluation of CBS gene expression through PCR, nucleic acid and protein blotting as well as enzyme assay; v) gross and microscopic examination of target tissues; vi) neurological, behavioral and developmental analysis of the resulting animals. In addition, the animal models will be made available to other laboratories conducting research on homocystinuria and PPAD/CeVD. These studies will elucidate disturbances of sulfur amino acid metabolism; further, they will permit the correlation of CBS deficiency and the clinical manifestation of disease and should facilitate the development of a biochemical rationale for treatment of certain mutant conditions.