A cell-specific inducible model of hearing loss

听力损失的细胞特异性诱导模型

• 批准号: 8004060
• 负责人: Albert Edge
• 金额: $ 19万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-15 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004060
• 关键词: A Mouse; Ablation; Address; Antibodies; Apoptotic; Auditory Physiology; Auditory system; Binding; Binding Sites; CMV promoter; Caspase; Caspase Gene; Cell Death; Cell Transplantation; Cell physiology; Cells; Cessation of life; Chemicals; Chimeric Proteins; Cochlea; Development; Dimerization; Ear; Engineering; Evaluation; Excision; FK506; Galactosidase; Ganglia; Generations; Genes; Genetic Models; Genetic Transcription; Genotype; Grant; Hair Cells; Histocompatibility Testing; Human; In Vitro; Individual; Knock-in Mouse; Labyrinth; LacZ Genes; Lead; Location; Measurement; Measures; Methods; Microinjections; Modeling; Molecular Profiling; Mus; Natural regeneration; Neurons; Oocytes; Organ; Pattern; Pharmaceutical Preparations; Plasmids; Procedures; Public Health; Sensorineural Hearing Loss; Site; Specificity; Structure; Tacrolimus Binding Proteins; Testing; Therapeutic; Time; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; analog; caspase-3; cell killing; cell type; deafness; gene cloning; genetic manipulation; hearing impairment; in vitro testing; in vivo; inner ear diseases; killings; monomer; mortality; mouse model; mutant; myristoylation; prevent; public health relevance; pup; recombinase; response; spiral ganglion; therapy development; transgene expression; vector

DESCRIPTION (provided by applicant): Deafness models have been made by a variety of approaches but problems with models in the mouse have hampered their use for studies of auditory physiology. Many methods rely on drugs that cause damage to cells in organs other than the ear and therefore lead to unacceptably high rates of mortality. The available methods also result in damage to multiple structures in the cochlea. Our objective is to develop a genetic model in which we can induce the death of specific cell types in the cochlea including hair cells and spiral ganglion neurons, two critical cell types for hearing loss. Such a model would expedite studies on cell function and development as well as inner ear regeneration. A model that was highly reproducible and that resulted in apoptotic cell death without damage to tissues that surround the dead cells would expedite procedures for cell replacement by cell transplantation or by use of genetic manipulation or therapeutic means of stimulating endogenous cells to replace the lost cell types. Our objective in Aim 1 is to make mice that contain a gene for cell targeting placed behind a CMV promoter for strong expression of the transgene. The construct contains a STOP sequence that prevents expression of a death-inducing caspase gene cloned between lox sites to permit its removal by a recombinase. In this construct the human caspase-3 gene is fused with FK506 binding sites, adding a further layer of control. Activity of the transgene will be tested in vitro and in vivo in Aim 1a. Our objective is to have lines of mice that provide partial or complete ablation, which is to be achieved by a transgenic approach in Aim 1b and a knock-in approach in Aim 1c. Our objective in Aim 2 is to test two lines of mice for targeted cell ablation in the cochlea. In the first line of mice we will target inner ear hair cells by crossing the transgenic to a Cre mouse specific for hair cells, while in the second line of mice we will cross the caspase transgenic with a Cre mouse that will allow targeting of spiral ganglion neurons. The approach, using a cross to a Cre mouse to target the cell type of choice, and treatment with an FK-506 analogue to induce cell death by dimerization of caspase-3, permits us to control both specification of tissue type and timing of cell death. PUBLIC HEALTH RELEVANCE: In this grant we will develop a mouse model to be used for the study of deafness. This model will be critical for the development of treatments for inner ear disorders in which replacement of hair cells or neurons will be evaluated. Because of the clear correlation between loss of cochlear cells and sensorineural hearing loss, improvements in our ability to regenerate these cells will lead directly to new treatments for loss of hearing.

描述（由申请人提供）：耳聋模型已通过多种方法制成，但小鼠模型的问题阻碍了其用于听觉生理学研究。许多方法依赖于对耳朵以外器官的细胞造成损害的药物，因此导致不可接受的高死亡率。可用的方法也导致耳蜗中多个结构的损伤。我们的目标是开发一种遗传模型，在该模型中，我们可以诱导耳蜗中特定细胞类型的死亡，包括毛细胞和螺旋神经节神经元，这是听力损失的两种关键细胞类型。这样的模型将加快对细胞功能和发育以及内耳再生的研究。高度可重复的并且导致凋亡性细胞死亡而不损伤死亡细胞周围的组织的模型将加速通过细胞移植或通过使用刺激内源性细胞以替代丢失的细胞类型的遗传操作或治疗手段进行细胞替代的程序。我们的目标1是制备含有置于CMV启动子后的细胞靶向基因的小鼠，用于转基因的强表达。该构建体含有阻止在lox位点之间克隆的诱导死亡的半胱天冬酶基因的表达的终止序列，以允许其通过重组酶去除。在该构建体中，人胱天蛋白酶-3基因与FK 506结合位点融合，增加了另一层对照。将在Aim 1a中体外和体内测试转基因的活性。我们的目标是获得提供部分或完全消融的小鼠品系，这将通过Aim 1b中的转基因方法和Aim 1c中的敲入方法来实现。我们在目标2中的目标是测试两个品系的小鼠用于耳蜗中的靶向细胞消融。在第一系小鼠中，我们将通过将转基因小鼠与毛细胞特异性的Cre小鼠杂交来靶向内耳毛细胞，而在第二系小鼠中，我们将半胱天冬酶转基因小鼠与Cre小鼠杂交，这将允许靶向螺旋神经节神经元。该方法使用与Cre小鼠杂交以靶向选择的细胞类型，并用FK-506类似物处理以通过半胱天冬酶-3的二聚化诱导细胞死亡，使我们能够控制组织类型的规格和细胞死亡的时间。 公共卫生相关性：在这项资助中，我们将开发一种用于耳聋研究的小鼠模型。该模型对于开发内耳疾病的治疗方法至关重要，其中将评估毛细胞或神经元的替代。由于耳蜗细胞的损失和感觉神经性听力损失之间存在明显的相关性，我们再生这些细胞的能力的提高将直接导致听力损失的新治疗方法。