TARGETED OVEREXPRESSION OF PTH RELATED PEPTIDE

小鼠基因操作实验表明，甲状旁腺激素相关蛋白（PTHrP）是一种发育调控分子。例如，PTHrP在角化细胞、乳腺上皮细胞和软骨细胞中的过度表达会导致每种情况下的发育表型，而敲除该基因会导致出生时致命的软骨营养不良。我们通过遗传策略挽救了PTHrP-null，并且该小鼠提供了以前未被发现的PTHrP在具有共同上皮-间质形态发生的组织中的作用的窗口。这种表型包括乳腺上皮发育和牙齿出疹的失败以及皮肤的多种异常。通过角蛋白-14-PTHrP转基因将PTHrP传递到这些上皮部位可挽救这些发育不良特征。我们最近也在pthrp缺失的中枢神经系统中发现了一种与年龄相关的神经退行性过程，并且似乎以兴奋毒性为基础。在这里，我们建议创建一系列小鼠模型，以便在体内更详细地研究这些调节作用。目的1。我们将在角蛋白14启动子的控制下创建“脱开”和“脱开”系统，以提供增殖上皮中PTHrP表达的调节。这将使我们能够研究PTHrP对胚胎和青春期乳腺发育阶段分支形态发生的影响，试图将PTHrP与牙齿运动/破骨细胞吸收的多次迭代联系起来，并探讨皮肤中PTHrP缺乏是否与早衰有关的问题。目标2。我们将使用同源重组将lacZ报告基因插入PTHrP等位基因中，并将产生携带该报告基因作为PTHrP空等位基因之一的PTHrP空小鼠。这将使研究基因的时空表达具有以前不可能的灵敏度和保真度，一个恰当的例子是在Aim 3中提出使用这种小鼠。目标3。我们的工作假设是PTHrP在一个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），因此神经元可以对抗长潜伏期兴奋性毒性。lacZ报告基因将能够高分辨率地研究lacZ/PTHrP缺失的双杂合子的神经退行性过程，我们将尝试在泛神经元启动子（神经元特异性烯醇化酶）的控制下，通过PTHrP转基因从基因上挽救或逆转这一过程。

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).