TARGETED OVEREXPRESSION OF PTH RELATED PEPTIDE

小鼠基因操作实验表明，甲状旁腺相关蛋白（PTHrP）作为一种发育调控分子。例如，PTHrP在角质形成细胞、乳腺上皮细胞和软骨细胞中的过表达在每种情况下导致发育表型，而基因敲除导致出生时致命的软骨营养不良。 我们已经通过遗传策略拯救了PTHrP无效，这只小鼠提供了一个窗口，以前不受重视的PTHrP在组织中的作用，共享一个共同的上皮间充质形态发生。 这种表型包括乳腺上皮发育和牙齿萌出的失败以及皮肤的多种异常。 通过角蛋白-14-PTHrP转基因将PTHrP递送到这些上皮部位可以挽救这些发育异常特征。 我们最近还确定了一个神经退行性过程中的PTHrP无效的中枢神经系统，这是年龄相关的，似乎有兴奋性毒性作为其基础。 在这里，我们建议建立一系列的小鼠模型，使这些调节作用在体内进行更详细的研究。目标1. 我们将在角蛋白-14启动子的控制下创建“tet-off”和“tet-on”系统，以提供增生上皮中PTHrP表达的调节。 这将使我们能够研究PTHrP对乳腺发育的胚胎期和青春期阶段的分支形态发生的影响，试图将PTHrP与牙齿移动/骨吸收的多次迭代联系起来，并探讨皮肤中PTHrP缺乏是否与过早衰老有关的问题。目标二。 我们将使用同源重组将lacZ报告基因插入PTHrP等位基因中，并将产生携带报告基因作为PTHrP无效等位基因之一的拯救的PTHrP无效小鼠。 这将使得能够以以前不可能的灵敏度和保真度研究基因的时空表达，一个恰当的例子是在目标3中提议使用这种小鼠。目标3： 我们的工作假设是PTHrP在自我保护反馈回路中起作用（去极化产生PTHrP产生1型受体产生L型钙通道的抑制），从而神经元可以对抗长潜伏期兴奋性毒性。lacZ报告将使lacZ/PTHrP无效双杂合子的神经退行性过程的高分辨率研究，我们将试图拯救/逆转这一过程的遗传通过PTHrP转基因控制下的泛神经元启动子（神经元特异性烯醇化酶）。

Gene manipulation experiments in mice indicate that parathyroid hormone-related protein (PTHrP) functions as a developmental regulatory molecule. For example, overexpression of PTHrP in keratinocytes, mammary epithelial cells and chondrocytes results in a developmental phenotype in each case, while knockout of the gene leads to a chondrodystrophy that is lethal at birth. We have rescued the PTHrP-null via a genetic strategy, and this mouse provides a window on previously unappreciated PTHrP effects in tissues that share a common epithelial-mesenchymal morphogenesis. This phenotype includes failures in mammary epithelial development and tooth eruption and multiple abnormalities in skin. Delivery of PTHrP to these epithelial sites via a keratin-14-PTHrP transgene rescues these dysplastic features. We have also recently identified a neurodegenerative process in the PTHrP-null CNS that is age-related and seems to have excitotoxicity as its basis. Here we propose to create a series of mouse models to enable more detailed study of these regulatory effects in vivo. Aim 1. We will create "tet-off" and "tet-on" systems under the control of the keratin-14 promoter to provide regulated PTHrP expression in proliferative epithelia. This will allow us to study PTHrP effects on branching morphorgenesis during both embryonic and adolescent phases of mammary development, attempt to implicate PTHrP in multiple iterations of tooth movement/osteoclastic resorption, and explore the question of whether PTHrP deficiency in skin is associated with premature aging. Aim 2. We will use homologous recombination to insert a lacZ reporter gene into a PTHrP allele and will generate rescued PTHrP-null mice bearing the reporter as one of the PTHrP-null alleles. This will enable the study of the temporospatial expression of the gene with a sensitivity and fidelity not previously possible, an apt example being the proposed use of this mouse in Aim 3. Aim 3. It is our working hypothesis here that PTHrP functions in an autoprotective feedback loop (depolarization yields PTHrP yields type 1 receptor yields inhibition of L-type calcium channels) whereby neurons can combat long-latency excitotoxicity. The lacZ reporter will enable high resolution study of the neurodegenerative process in lacZ/PTHrP-null double heterozygotes, and we will attempt to rescue/reverse this process genetically via a PTHrP transgene under the control of a pan-neuronal promoter (neuron-specific enolase).