Knock-In Model for Inner Ear Hair Cell Regeneration

描述（由申请人提供）：感觉毛细胞（HC）的丧失是人类耳聋、耳炎和平衡障碍的主要原因。然而，尽管许多实验室取得了很大的进展，毛细胞发育和再生的分子基础仍然不清楚。我们提出验证新的假设，即如果我们能够诱导hc重新表达至关重要的生长因子骨形态发生蛋白4 (BMP4)，哺乳动物毛细胞再生将是可能的。在这里提出的实验中，我们将制作一种转基因“敲入”小鼠，使BIVIP4在双控制系统下可以在哺乳动物毛细胞中表达。我们将设计构建一个全长BMP4在毛细胞特异性brn3.1启动子的控制下，在一个可诱导的系统中，为转基因小鼠提供多西环素（DOX），使BMP4在通常不表达BIVIP4的内耳终末分化毛细胞中特异性表达。BIVIP4是一种轴向模式形态形成因子，但更重要的是，它是一种主要的“可塑性”基因，使感觉细胞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天的永生化小鼠耳囊的内耳细胞系和培养的小鼠胚胎干细胞（ES）中表达这种构建体。我们已经构建了驱动荧光素酶的结构，来测试原理的证明。我们还可以比较用DOX诱导的细胞与未经RT-PCR和基因阵列技术诱导的细胞的基因表达库。接下来，我们将制造一种转基因敲入小鼠，它能够在内耳的所有毛细胞中重新表达BMP4。我们将研究存在和不存在DOX时这些小鼠的细胞形态和HC修复和再生能力；也就是说，当毛细胞能够或不能够重新表达BIVIP4时。我们正在测试一种假设，即如果BIVIP4在内耳毛细胞中重新表达，使这些细胞恢复到更早的“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暴露后再生感觉上皮。通过局部噪声暴露或精确剂量和递送氨基糖苷，我们可以改变病变的部位和程度，并确定修复、再生和/或保存在恢复功能感觉上皮中的作用程度。

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.