A cell-specific inducible model of hearing loss

描述（由申请人提供）：耳聋模型已通过多种方法制作，但小鼠模型的问题阻碍了它们在听觉生理学研究中的应用。许多方法依赖于药物，这些药物会对耳朵以外的器官细胞造成损害，因此导致令人无法接受的高死亡率。现有的方法也会对耳蜗的多个结构造成损伤。我们的目标是开发一种遗传模型，在这种模型中，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。这种模型将加速细胞功能和发育以及内耳再生的研究。如果一个模型具有高度可重复性，并能导致凋亡细胞死亡而不损害死亡细胞周围的组织，那么它将加快通过细胞移植或使用遗传操作或刺激内源性细胞替代丢失细胞类型的治疗手段来替代细胞的程序。我们在Aim 1中的目标是使含有细胞靶向基因的小鼠置于CMV启动子后面，以强表达转基因。该构建体包含一个STOP序列，该序列可阻止在lox位点之间克隆的致死caspase基因的表达，从而允许重组酶将其移除。在这个结构中，人类caspase-3基因与FK506结合位点融合，增加了进一步的控制层。转基因的活性将在体外和体内在Aim 1a中进行测试。我们的目标是获得提供部分或完全消融的小鼠系，这将通过Aim 1b的转基因方法和Aim 1c的敲入方法来实现。我们在Aim 2中的目标是测试两种小鼠系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与特定于毛细胞的Cre小鼠杂交来靶向内耳毛细胞，而在第二行小鼠中，我们将将caspase转基因与Cre小鼠杂交，从而可以靶向螺旋神经节神经元。该方法使用与Cre小鼠的杂交来靶向选择的细胞类型，并使用FK-506类似物通过caspase-3的二聚化诱导细胞死亡，使我们能够控制组织类型和细胞死亡时间的规格。

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.