TARGETED OVEREXPRESSION OF PTH-RELATED PEPTIDE

描述(改编自申请人摘要)：PTH和PTHrP 基因是编码调节分子的一个小家族的成员。 它们的功能似乎截然不同。PTHrP基因是 显然是体内现代靶向策略的理想候选者，以及 最近应用了两种这样的策略，这两种策略都揭示了 有趣的表型。 尽管这两个战略涉及镜像方法(有针对性 一方面是基因破坏，另一方面是有针对性的过度表达， 其他)在两个实验中发现的甲状旁腺素受体功能(S)很可能是 基本上是一样的。本申请提出了四个有针对性 过度表达策略涉及皮肤、乳房、子宫和 软骨。每个实验的重点(以及作为一个 整个)正在体内工作，只需要有限的资源 体外衍生实验。首先，人角蛋白-14(K14) 启动子已经被用来将PTHrP的过度表达靶向表皮 产生一种表型，似乎反映了初生代 毛囊启动失败。因此，该应用程序建议： A)充分描述K14-PTHrP小鼠的特征；b)研究自分泌/旁分泌 有限数量的体外实验中的信号机制；以及c) 靶向甲状旁腺素(1-84)，以及嵌合和/或缺失的分子 目的：研究PTHrP在体内的结构与功能关系。 其次，K14-PTHrP小鼠似乎也表现出在 小叶肺泡乳房发育，可能反映了 K14在乳腺肌上皮细胞中的启动子。应用程序 建议：a)充分描述K14-PTHrP的乳腺缺陷 小鼠；以及b)使用MMTV将PTHrP过表达靶向乳房 同基因小鼠中的启动子/增强子；这种方法旨在允许 激素调控MMTV-PTHrP的表达，也允许乳房 上皮脂肪垫移植定位甲状旁腺素相关蛋白的实验研究 发育影响/缺陷。第三，申请提出了 靶向PTHrP在小鼠子宫肌层和子宫内膜中的过表达 使用猪子宫铁蛋白启动子。相关的实验将 重点放在：a)推测甲状旁腺素受体参与分娩过程 以及b)一氧化氮在介导甲状旁腺素受体活性中的作用。 子宫肌层。第四，应用程序提出以PTHrP为目标 高表达对增殖期和增生期软骨细胞的影响 鼠II型胶原启动子(原-α-L(11))。这些 实验将集中在：a)明显的甲状旁腺素受体功能(S)在 软骨细胞分化的发育程序；和b)一个交叉- 试图逆转或挽救的繁育互补试验 PTHrP基因靶向(PTHrP缺失)小鼠的表型。

DESCRIPTIONS (Adapted from the Applicant's Abstract): The PTH and PTHrP genes are members of a small family that encode regulatory molecules whose functions seem to be remarkably different. The PTHrP gene is clearly an ideal candidate for modern targeting strategies in vivo, and two such strategies have recently been applied, both revealing very interesting phenotypes. Although these two strategies involved mirror-image approaches (targeted gene disruption, on the one hand, and targeted overexpression, on the other) the PTHrP function(s) uncovered in both experiments may well be fundamentally the same. The present application proposes four targeted overexpression strategies involving the skin, breast, uterus, and cartilage. The emphasis of each experiment (and of the proposal as a whole) is on work in vivo, and only limited resources are requested for derivative experiments in vitro. First, the human keratin-14 (K14) promoter has been used to target PTHrP overexpression to the epidermis generating a phenotype that appears to reflect a primary failure of hair follicle initiation. The application thus proposes to: a) fully characterize the K14-PTHrP mice; b) study autocrine/paracrine signaling mechanisms in a limited number of experiments in vitro; and c) target PTH (1-84), as well as chimeric and/or deleted molecules, to skin in order to study PTHrP structure-function relationships in vivo. Second, the K14-PTHrP mice appear also to manifest a failure in lobuloalveolar mammary development, possibly reflecting activity of the K14 promoter in myoepithelial cells in the breast. The application proposes: a) to characterize fully the mammary defect in the K14-PTHrP mice; and b) to target PTHrP overexpression to the breast using the MMTV promoter/enhancer in syngeneic mice; this approach is intended to permit hormonal manipulation of MMTV-PTHrP expression, and also allow mammary epithelial fat pad transplantation experiments in order to pinpoint PTHrP developmental effects/defects. Thirdly, the application proposes to target PTHrP overexpression to the mouse myometrium and endometrium using the porcine uteroferrin promoter. The associated experiments will focus on:a) putative PTHrP involvement in the process of parturition per se; and b) the role of nitric oxide in mediating PTHrP actions in the myometrium. Fourthly, the application proposes to target PTHrP overexpression to proliferative and prehypertrophic chondrocytes using the mouse type II collagen promoter (pro-alpha-l(ll)). These experiments will focus on: a) apparent PTHrP function(s) in the developmental program of chondrocyte differentiation; and b) a cross- breeding complementation experiment in attempt to reverse or salvage the phenotype in the PTHrP gene-targeted (PTHrP null) mice.