Knock-In Model for Inner Ear Hair Cell Regeneration

描述（由申请人提供）：感觉毛细胞（HC）损失是人类耳聋、耳鸣和平衡障碍的主要原因。然而，尽管许多实验室取得了很大进展，毛细胞发育和再生的分子基础仍然不清楚。我们建议测试新的假设，即哺乳动物毛细胞再生在体内将是可能的，如果我们可以诱导毛细胞重新表达至关重要的生长因子骨形态发生蛋白4（BMP 4）。在这里提出的实验中，我们将制作一个转基因“敲入”小鼠，其中BIVIP 4可以在双重控制系统下在哺乳动物毛细胞中表达。我们将在tet-on诱导系统中，将全长BMP 4置于毛细胞特异性brn3.1启动子的控制下，从而为转基因小鼠提供强力霉素（DOX）将特异性地在通常不表达BIVIP 4的内耳终末分化毛细胞中启动BMP 4表达。BIVIP 4是一种轴向模式化形态原，但更重要的是，它是一种主要的“可塑性”基因，使感觉和神经元细胞保持增殖状态，类似于干细胞。我们将首先在来源于第9天Immortomouse耳囊的永生化内耳细胞系和培养的小鼠胚胎干细胞（ES）中表达这种tet-on构建体。我们已经构建了驱动荧光素酶的结构，以测试原理的证明。我们还可以比较用DOX诱导的细胞的基因表达谱与未通过RT-PCR和基因阵列技术诱导的细胞的基因表达谱。接下来，我们将制造一种能够在内耳的所有毛细胞中重新表达BMP 4的转基因敲入小鼠。我们将研究这些小鼠在存在和不存在DOX的情况下的细胞形态和HC修复和再生能力;也就是说，当毛细胞能够重新表达BIVIP 4时。我们正在检验这样一个假设，即如果BIVIP 4在内耳毛细胞中重新表达，使这些细胞恢复到早期的干细胞样状态，我们将能够在噪声诱导的创伤或耳毒性氨基糖苷类暴露后再生感觉上皮。通过使用局灶性噪声暴露或精确给药和递送氨基糖苷类，我们可以改变病变的部位和程度，并确定修复、再生和/或保存在恢复功能性感觉上皮中发挥作用的程度。

DESCRIPTION (provided by applicant): Sensory hair cell (HC) loss is a major cause of human deafness, tinnitis and balance disorders. However, despite great progress in many laboratories, the molecular bases for both hair cell development and regeneration are still not understood. We propose to test the novel hypothesis that mammalian hair cell regeneration in vivo will be possible if we can induce HCs to re-express the critically important growth factor Bone Morphogenetic Protein 4 (BMP4). In the experiments proposed here, we will make a transgenic "knock-in" mouse, in which BIVIP4 can be expressed in mammalian hair cells under a dual control system. We will engineer constructs that place a full-length BMP4 under the control of the hair-cell specific brn3.1 promoter, in a tet-on inducible system, so that providing the transgenic mice with doxycycline (DOX) will turn on BMP4 expression specifically in terminally differentiated hair cells of the inner ear that normally do not express BIVIP4. BIVIP4 is an axial patterning morphogen, but more significantly, it is a major "plasticity" gene that keeps sensory and neuronal cells in a proliferative state, resembling stem cells. We will first express this tet-on construct in immortalized inner ear cell lines derived from day 9 Immortomouse otocysts and in mouse embryonic stem cells (ES) in culture. We have made constructs that drive luciferase, to test the proof of principle. We can also compare the genetic expression repertoire of the cells induced with DOX to those of cells that are not induced by RT-PCR and by gene array techniques. Next we will make a transgenic knock-in mouse that is capable of re-expressing BMP4 in all of the hair cells of the inner ear. We will study the cellular morphology and HC repair and regenerative capacity of these mice in the presence and absence of DOX; that is, when the hair cells are either capable of re-expressing BIVIP4 or not. We are testing the hypothesis that if BIVIP4 is re-expressed in hair cells of the inner ear, returning these cells to an earlier more 11 stem cell-like" state, we will be able to regenerate a sensory epithelium after either noise-induced trauma or ototoxic aminoglycoside exposure. By using focal noise exposure or precisely dosed and delivered aminoglycosides, we can vary the site and extent of the lesions, and determine the degree to which repair, regeneration and/or preservation play a role in restoring a functional sensory epithelium.