TARGETED OVEREXPRESSION OF PTH-RELATED PEPTIDE

说明（改编自申请人的摘要）：PTH和PTHrP 基因是一个小家族的成员， 它们的功能似乎截然不同。 PTHrP基因是 显然是现代体内靶向策略的理想候选者， 最近已经应用了两种这样的策略，它们都揭示了 有趣的表型 虽然这两项战略涉及镜像方法（针对 一方面是基因破坏，另一方面是靶向过表达， 其他）在两个实验中发现的PTHrP功能很可能是 基本上是一样的。 本申请提出了四个目标， 过度表达策略涉及皮肤、乳腺、子宫， 软骨 每个实验的重点（以及作为一个 整个项目）都是在体内开展工作，只要求有限的资源， 体外衍生实验。 人类角蛋白-14（K14） 启动子已被用于将PTHrP过表达靶向表皮 产生一种表现型， 毛囊起始失败。因此，申请书建议： a）充分表征K14-PTHrP小鼠; B）研究自分泌/旁分泌 在有限数量的体外实验中的信号传导机制;以及c） 将PTH（1-84）以及嵌合和/或缺失分子靶向皮肤 研究PTHrP在体内的结构与功能关系。 第二，K14-PTHrP小鼠似乎也表现出在以下方面的失败： 小叶肺泡乳腺发育，可能反映了 乳腺肌上皮细胞中的K14启动子。 应用 建议：a）充分表征K14-PTHrP中的乳腺缺陷 小鼠;和B）使用MMTV将PTHrP过表达靶向乳房 启动子/增强子在同基因小鼠中的表达;这种方法旨在允许 MMTV-PTHrP表达的激素操纵，并且还允许乳腺 上皮脂肪垫移植实验，以确定PTHrP 发育的影响/缺陷。 第三，申请书建议 PTHrP在小鼠子宫肌层和子宫内膜的靶向过表达 使用猪子宫铁蛋白启动子。 相关实验将 重点：a）假定PTHrP参与分娩过程， se;和B）一氧化氮在介导PTHrP作用中的作用， 子宫肌层 第四，本申请提出靶向PTHrP 过度表达到增殖和前肥大软骨细胞， 小鼠II型胶原蛋白启动子（pro-alpha-1（II））。 这些 实验将集中于：a）在所述组织中的表观PTHrP功能， 软骨细胞分化的发育程序;和B）交叉- 育种互补实验，试图扭转或挽救 PTHrP基因靶向（PTHrP无效）小鼠中的表型。

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.