DEVELOPMENT OF A MODEL FOR TAY-SACHS DISEASE

泰-萨二氏病，一种主要影响 神经系统，已在分子水平上进行了表征， 与酶的缺陷或异常α链有关 氨基己糖苷酶（氨基己糖苷酶A）。 基因编码突变 已经鉴定出氨基己糖苷酶A（HEXA）在人体中导致 婴儿期、少年期或成人期发病形式的发展 泰-萨克斯病 开发新的治疗干预方法， 由于缺乏足够的动物， 评估新治疗方法疗效的模型， 包括基因治疗。 修饰小鼠特定基因的能力 利用胚胎干细胞通过同源重组，结合 修饰后的胚胎干细胞能够促进 胚胎干细胞-囊胚嵌合体中生殖细胞的研究开辟了一个新的领域 用来制作人类疾病的动物模型。 因此 本申请提出开发婴儿的小鼠模型， 青少年和成人型泰-萨二氏病通过一种新的同源 重组过程。 本申请的总体目标包括：1）开发 通过HEXA失活的婴儿Tay-Sachs病小鼠模型 ES细胞中的基因。 基因的失活将通过以下方式完成： 导致插入neo的同源重组事件， hsv-tk基因外显子6; 2）开发一种新的同源 在特定区域引入微小突变的重组程序 HEXA基因。 这种新的程序，命名为“击中和开关”，将是 用于在位置269处引入Gly至Ser氨基酸取代 在HEXA基因的外显子7中。 一旦被修饰，ES细胞将被用于 产生成人型泰-萨二氏病的小鼠模型; 3）利用 “命中和转换”同源重组程序，以引入Gly 在外显子7中的位置250处替换为Asp氨基酸。 动物 外显子7修饰的纯合子将作为青少年的模型。 Tay-Sachs; 4）通过光和 电子显微镜检查的类型，严重程度和时间 神经元异常的表现。 此外， 基因修饰对氨基己糖苷酶A活性的影响，以及对积累的影响 将确定神经系统中单个神经节苷脂的含量。

Tay-Sachs disease, a lysosomal storage disease affecting primarily the nervous system, has been characterized at the molecular levels and found to be associated with a deficient or abnormal alpha chain of the enzyme hexosaminidase (hexosaminidase A). Mutations in the gene encoding hexosaminidase A (HEXA) have been identified that, in humans, result in the development of the infantile, juvenile, or the adult onset form of Tay-Sachs disease. The development of new methods of therapeutic intervention to alleviate this devastating disease has been hampered by the lack of adequate animal models on which to evaluate the efficacy of new treatment methods, including gene therapy. The ability to modify specific genes in mouse embryonic stem cells by the use of homologous recombination, combined with the ability of the modified ES cells to contribute to the formation of the germ cells in ES-blastocyst chimeras, has opened up a new venue with which to generate animal models of human diseases. Accordingly, the current application proposes to develop a mouse model of infantile, juvenile, and adult-onset Tay-Sachs disease by a novel homologous recombination procedure. The overall objectives of this application include: 1) development of a mouse model of infantile Tay-Sachs disease by inactivation of the HEXA gene in ES cells. The inactivation of the gene will be accomplished by a homologous recombination event resulting in insertion of the neo and hsv-tk genes in exon 6; 2) development of a novel homologous recombination procedure to introduce subtle mutations in specific region of the HEXA gene. This novel procedure, named "hit and switch", will be used to introduce a Gly to Ser amino acid substitution at position 269 in exon 7 of the HEXA gene. Once modified, ES cells will be used to generate a mouse model of adult-onset Tay-Sachs; 3) utilization of the "hit and switch" homologous recombination procedure to introduce an Gly to Asp amino acid substitution at position 250 in exon 7. Animals homozygous for the exon 7 modification will serve as models of juvenile Tay-Sachs; 4) characterization of generated animal models by light and electron microscopy in terms of type, degree of severity, and the timing of appearance of neuronal abnormalities. Additionally, the effects of gene modification on hexosaminidase A activity, and on the accumulation of individual gangliosides in the nervous system will be determined.