DEVELOPMENT OF A MODEL FOR TAY-SACHS DISEASE

泰萨克斯病模型的开发

• 批准号: 2269095
• 负责人: Jorge A Piedrahita
• 金额: $ 11.26万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2269095
• 关键词: Tay Sachs disease; beta N acetylhexosaminidase; disease /disorder model; embryonic stem cell; enzyme induction /repression; laboratory mouse; model design /development; recombinant DNA

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.

Tay-Sachs病，一种溶酶体储存疾病，主要影响 神经系统已在分子水平上进行表征，发现 与酶的不足或异常α链有关 己糖胺酶（己糖胺酶A）。 基因编码中的突变 己糖胺酶A（Hexa）已被确定，在人类中， 婴儿，少年或成人发作形式的发展 Tay-Sachs病。 开发新的治疗干预方法以减轻 由于缺乏足够的动物，这种毁灭性疾病受到了阻碍 评估新治疗方法有效性的模型， 包括基因疗法。 修改小鼠特定基因的能力 胚胎干细胞通过使用同源重组，合并 具有修饰的ES细胞有助于形成的能力 Es-blastocyst嵌合体中的生殖细胞已经打开了一个新的场地 用它产生人类疾病的动物模型。 因此， 当前的应用程序建议开发婴儿的小鼠模型， 新型同源物 重组程序。 此应用程序的总体目标包括：1）开发 通过灭活Hexa的婴儿TAY-SACHS疾病的小鼠模型 ES细胞中的基因。 基因的失活将通过 同源重组事件，导致插入NEO和 外显子6中的HSV-TK基因； 2）开发新颖的同源性 重组程序以引入特定区域的细微突变 Hexa基因。 这个名为“ hit and switch”的新颖过程将是 用于在269的位置引入GLY氨基酸取代 在Hexa基因的第7外显子中。 一旦修改，ES单元将用于 生成成人发作的小鼠模型； 3）利用 “命中和切换”同源重组程序，以引入gly 在外显子7中250的位置上取代ASP氨基酸。 外显子7修饰的纯合子将用作少年的模型 Tay-Sachs； 4）通过光和 电子显微镜在类型，严重程度和时机方面 神经元异常的外观。 另外， 己糖胺酶A活性和积累的基因修饰 神经系统中的单个神经毒剂将被确定。