A cell-specific inducible model of hearing loss

听力损失的细胞特异性诱导模型

基本信息

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
项目类别：
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

项目摘要

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）：已经通过多种方法制作了耳聋模型，但是鼠标中的模型问题阻碍了其用于听觉生理学的研究。许多方法依赖于对耳朵以外的器官中其他细胞损害的药物，因此导致死亡率高得多。可用的方法还导致对耳蜗中多个结构的损害。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，两种关键细胞类型的听力损失。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。高度可重现的模型，导致凋亡细胞死亡，而不会损害围绕死细胞的组织，这将加快通过细胞移植或使用遗传操纵或刺激内源性细胞替代丢失细胞类型的遗传操纵或治疗手段来代替细胞的程序。我们目标1的目标是使含有用于细胞靶向基因的基因，以置于CMV启动子后方，以强烈表达转基因。该构建体包含一个停止序列，该序列可防止诱发死亡的caspase基因在Lox位点之间克隆的caspase基因，从而允许其通过重组酶去除。在此构造中，人caspase-3基因与FK506结合位点融合，并增加了一层控制层。转基因的活性将在AIM 1A中在体外和体内进行测试。我们的目标是拥有提供部分或完整消融的小鼠线，这是通过AIM 1B中的转基因方法和AIM 1C中的敲门方法来实现的。我们目标2的目标是测试两条小鼠在耳蜗中的靶向细胞消融。在第一行小鼠中，我们将通过将转基因跨越针对毛细胞的CRE小鼠来瞄准内耳毛细胞，而在第二行小鼠中，我们将使用CRE小鼠穿越caspase转基因，该小鼠将允许靶向螺旋神经神经元。该方法是使用十字与CRE小鼠靶向选择细胞类型的方法，并用FK-506模拟方法处理通过caspase-3二聚化诱导细胞死亡，使我们能够控制组织类型的规范和细胞死亡的时间。公共卫生相关性：在这笔赠款中，我们将开发一种鼠标模型，用于研究耳聋。该模型对于将评估毛细胞或神经元替代的内耳疾病的治疗方法至关重要。由于耳蜗细胞的丧失与感官性听力丧失之间存在明显的相关性，因此我们再生这些细胞的能力的提高将直接导致新的治疗方法，以造成听力丧失。