Inducible Expression of Exogenous Potassium Channels in Hair Cells

毛细胞中外源钾通道的诱导表达

• 批准号: 8129901
• 负责人: JEFFREY R HOLT
• 金额: $ 21.69万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8129901
• 关键词: Address; Affect; Aging; Animals; Antibodies; Auditory; Binding; Biochemical; Biological Assay; Biology; Birth; CMV promoter; Cell membrane; Cell physiology; Cells; Chemicals; Code; Culture Media; DNA Sequence; Development; Disease; Embryo; Engineering; Equilibrium; Excision; Exposure to; Functional disorder; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Hair Cells; Harvest; Hearing; Hearing problem; Human; Inherited; Inner Hair Cells; Isopropyl Thiogalactoside; Labyrinth; Lactose; Lead; Life; Life Cycle Stages; Longevity; Measures; Membrane Potentials; Messenger RNA; Modeling; Molecular; Mus; Myosin ATPase; Outer Hair Cells; Pattern; Physiological; Potassium; Potassium Channel; Protein Binding; Protein Biosynthesis; Proteins; Recovery of Function; Repression; Research; Research Project Grants; Rest; Role; Sampling; Sensory Hair; Shunt Device; Signal Transduction; Stimulus; System; Techniques; Time; Transgenic Mice; Transgenic Organisms; Type II Hair Cell; Vestibular Hair Cells; Western Blotting; Work; afferent nerve; age related; analog; auditory pathway; cell type; deafness; drinking water; equilibration disorder; experience; in vivo; interest; mouse model; novel; novel strategies; promoter; protein expression; receptor; response; seal; sound; synaptogenesis; tool; vestibular pathway; voltage; voltage clamp

DESCRIPTION (provided by applicant): The goal of this R21 research project is to develop and characterize a new mouse model that will be used to silence auditory and vestibular hair cell activity. We will generate a transgenic mouse line that allows for inducible, hair-cell expression of TREK1 potassium channels; also know as KCNK2 or K2P2.1 channels. Because TREK1 channels are open at rest and are not voltage-gated their expression will introduce an electrical shunt into the hair cell membrane which will effectively "clamp" the hair cell near the potassium equilibrium potential, about -80 mV. This simple model will render hair cells electrically silent, unable to perform voltage-dependent functions. The mouse model will be used to investigate, in a manner not available by other techniques, a number of functions in the auditory and vestibular systems that depend on the hair cell membrane potential. We plan to use the model to examine the role of the outer hair cell membrane potential in cochlear amplification, the role of spontaneous activity in synaptogenesis at the hair cell / 8th nerve afferent junction, the role of stimulus-evoked activity in the development and maturation of the auditory and vestibular systems. We will also use this system to investigate experience-dependent plasticity in a mouse model of age- related hearing and balance dysfunction, as well as during recovery of function. To generate our novel mouse model we will use a form of the bacterial Lac operator / repressor system that has been modified for use in mice (Cronin et al., 2001). We will introduce Lac operators into the Myosin7a promoter. The Myo7a promoter is active in hair cells throughout the animal's lifespan beginning as early as embryonic day 12 (Boeda et al., 2001). The modified Myo7a promoter will be inactive in the presence of Lac I protein. Simply by addition of the inducer, a nontoxic lactose analog, (IPTG), repression will be relieved and expression of TREK1 will commence. Therefore, using a simple chemical switch we will be able to turn on or off hair cell activity at anytime point during the lifespan of the mouse. We feel this work will be important for understanding the contribution of the receptor potential to a number of critical hair-cell functions. Development of this novel inducible system will also provide a powerful tool that can be modified to control hair-cell expression for any exogenous gene of interest. As such, this new tool will allow us to address several long standing questions regarding the basic biology of auditory and vestibular function and may also be developed into novel strategies for investigating hearing and balance disorders, which collectively affect >250 million people worldwide. PUBLIC HEALTH RELEVANCE: Sensory hair cells of the inner ear are critical for conversion of sound and balance information into electrical signals but unfortunately are susceptible to both inherited and acquired diseases that lead to deafness and balance problems. To understand how hair-cell disease leads to inner ear dysfunction we will use genetic tools to engineer a mouse that will allow us to turn hair cell function on and off at any time during the mouse's life. This mouse will provide a valuable new tool for studying deafness and balance disorders present at birth as well as acquired forms that appear later in life.

描述（由申请人提供）：该R21研究项目的目标是开发和表征一种新的小鼠模型，该模型将用于沉默听觉和前庭毛细胞活动。我们将产生一个转基因小鼠系，允许诱导，毛细胞表达TREK 1钾通道;也称为KCNK 2或K2P2.1通道。因为TREK 1通道在静止时是开放的，并且不是电压门控的，所以它们的表达将向毛细胞膜中引入电分流，这将有效地“钳位”接近钾平衡电位（约-80 mV）的毛细胞。这个简单的模型将使毛细胞电沉默，无法执行电压依赖的功能。小鼠模型将被用来调查，在一种方式不可用于其他技术，在听觉和前庭系统中的一些功能，依赖于毛细胞膜电位。我们计划使用该模型来检查耳蜗放大的外毛细胞膜电位的作用，在毛细胞/第8神经传入连接的突触发生的自发活动的作用，刺激诱发的活动在听觉和前庭系统的发育和成熟的作用。我们还将使用该系统来研究与年龄相关的听力和平衡功能障碍的小鼠模型以及功能恢复期间的经验依赖性可塑性。 为了产生我们的新型小鼠模型，我们将使用一种形式的细菌Lac操纵子/阻遏子系统，其已经被修饰用于小鼠（Cronin et al.，2001年）的报告。我们将Lac操纵子引入Myosin 7a启动子。Myo 7a启动子在早在胚胎第12天开始的整个动物寿命中在毛细胞中是有活性的（Boeda等人，2001年）的报告。修饰的Myo 7a启动子在Lac I蛋白存在下将是无活性的。简单地通过添加诱导剂，一种无毒的乳糖类似物（IPTG），阻遏将被解除，TREK 1的表达将开始。因此，使用一个简单的化学开关，我们将能够在小鼠生命周期的任何时候打开或关闭毛细胞的活动。 我们认为这项工作对于理解受体电位对一些关键毛细胞功能的贡献将是重要的。这种新的诱导系统的发展也将提供一个强大的工具，可以修改控制毛细胞表达的任何外源基因的兴趣。因此，这种新工具将使我们能够解决有关听觉和前庭功能的基础生物学的几个长期存在的问题，也可能发展成为研究听力和平衡障碍的新策略，这些疾病共同影响全球超过2.5亿人。 公共卫生相关性：内耳的感觉毛细胞对于将声音和平衡信息转换为电信号至关重要，但不幸的是，它容易受到遗传和后天疾病的影响，导致耳聋和平衡问题。为了了解毛细胞疾病是如何导致内耳功能障碍的，我们将使用遗传工具来设计一只小鼠，使我们能够在小鼠一生中的任何时候打开和关闭毛细胞功能。这种小鼠将为研究出生时的耳聋和平衡障碍以及后来出现的后天形式提供有价值的新工具。