Generation of mice with inducible Cre expression in adult cochlear hair cells

描述(由申请者提供)：我们在小鼠发育和成年过程中控制内耳毛细胞的基因表达的能力，对于理解人类听力生理学和耳聋的病理学至关重要。我们实验室和其他许多实验室的最新进展表明，在发育中的小鼠毛细胞中，基因表达可以通过空间和时间控制的方式进行操纵。然而，一个关键的特征仍然难以捉摸--我们操纵基因表达的能力，特别是在成熟的内毛细胞(IHC)和外毛细胞(OHC)。在这里，我们建议发展转基因或敲击小鼠，在成熟的耳蜗毛细胞(目标1中的毛细胞；目标2中的间质细胞和毛细胞)中表达可诱导的CreERT2，这是CRE和雌激素受体(Creer)的有效融合。这些小鼠品系的特性和可用性将使听觉研究人员能够使成熟毛细胞中他们感兴趣的基因失活或激活。在美国国立卫生研究院的基因敲除小鼠计划(KOMP)中提出的在它们基因组中的每个小鼠基因的条件(LoxP)等位基因的创建，将为我们产生的可诱导的Cre系的使用提供补充资源。最后，我们在这里采用的策略可以用于在任何给定的时间在内耳的其他成熟细胞类型(即IHC、螺旋神经节、纹状体纤维细胞和边缘细胞)中特异性地表达基因。所有小鼠品系都将用129S7/C57BL/6J混合品系或FVB/NJ品系进行基因工程，转移到CBA/CAJ品系，然后保存在NIH赞助的突变小鼠区域资源中心(MMRRC)。公共卫生相关声明估计美国有2800万人是聋人或重听人。大约有150万3岁或以上的人双耳失聪，每1000名活产儿中就有2到3人患有先天性听力损失。美国有4000多万人遭受不同程度的噪音导致的听力损失。尽管我们对这些听力障碍的了解取得了重大进展，但人们对这些疾病的病因和成年人的正常听力过程知之甚少。许多遗传因素(基因)在突变时会导致人的听力障碍，对婴儿和成年人都起着关键作用。我们建议开发基因工具，使我们能够在成年动物模型中研究与听力相关的基因的功能。这些工具将能够在动物成年后的任何时间通过手术激活听力过程中一组特定细胞中的基因。这种时间和空间的精确度对于我们理解听力过程和开发干预听力障碍的治疗靶点至关重要。

DESCRIPTION (provided by applicant): Our ability to manipulate gene expression specifically in the hair cells of the inner ear during development and adulthood in mice is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances from our laboratory and many others have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. However, a key feature remains elusive -- our ability to manipulate gene expression specifically in mature inner hair cells (IHCs) and outer hair cells (OHCs). Here we propose to develop transgenic or knockin mice that express inducible CreERT2, an effective fusion of Cre and estrogen receptor (CreER), in mature cochlear hair cells (OHCs in Aim 1; IHCs and OHCs in Aim 2). The characterization and availability of these mouse lines will enable auditory researchers to inactivate or activate genes of their interest in mature hair cells. The creation of conditional (loxP) alleles of every mouse gene in their genome, which has been proposed in the NIH's Knockout Mouse Project (KOMP), will provide a complementary resource for the use of the inducible Cre lines that we generate. Finally, the strategies we adopt here can be used to express genes specifically in other mature cell types of the inner ear (i.e., IHCs, spiral ganglia, stria fibrocytes and marginal cells) at any given time. All mouse lines will be genetically engineered in 129S7/C57BL/6J mixed or FVB/NJ strains, transferred to the CBA/CaJ strain, and then deposited in the NIH-sponsored Mutant Mouse Regional Resource Center (MMRRC). Public Health Relevance Statement An estimated 28 million people in the United States are deaf or hard of hearing. Approximately 1.5 million individuals aged 3 years or older are deaf in both ears and 2 to 3 per 1,000 live births suffer congenital hearing loss. More than 40 million persons in the United States suffer various levels of noise induced hearing loss. Despite the significant progress in our understanding of these hearing disorders, very little is known about the disease causes and about the normal hearing processes in adults. Many genetic factors (genes) that, when mutated, cause hearing impairment in people, play critical roles in both infants and adults. We propose here to develop genetic tools that would allow us to investigate the function of hearing related genes in adult animal models. These tools will be able to surgically activate genes in a specific set of cells in the hearing process at any time of the adult life of the animal. This temporal and spatial precision is critical for our understanding of hearing process and developing therapeutic targets for intervention of the hearing disorders.