REGULATION OF GH BY A TETRACYCLINE-REPRESSIBLE SYSTEM

四环素抑制系统对 GH 的调节

• 批准号: 6198522
• 负责人: WILLIAM W MORGAN
• 金额: $ 7.23万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6198522
• 关键词: age difference; aging; animal breeding; biological models; biotechnology; blood tests; enzyme induction /repression; gene expression; genetic manipulation; genetic promoter element; genetic transcription; genetic translation; genetically modified animals; growth /development; insulinlike growth factor; laboratory mouse; longevity; model design /development; somatotropin; tetracyclines

The purpose is to develop and initially characterize a novel transgenic mouse model in which the circulating levels of GH are normal but can be titrated downwards using a tetracycline-repressible transactivators system. Aim 1 is to develop a knockout-knockin transgenic mouse model which will [1] knockout the endogenous murine GH gene and [2] insert a DNA construct which will place the expression of the tetracycline (Tet)- repressible transactivator protein (tTAr) under the control of the endogenous mouse GH promoter. The transactivator will in turn activated a minimal transactivator-dependent (TD) promoter to drive the expression of the rat GH gene (rGH). With this strategy the endogenous GH promoter will essentially drive the expression of rat GH. Since all of the normal regulatory mechanisms will be intact, GH levels should approximately the normal levels found in the mouse and should show the pulsatile release characteristic of this hormone. Aim 2 will characterize and validate the model by first determining how well the knockout- knockin model recapitulates the normal secretion of GH. Plasma GH and insulin-like growth factor (IGF-1) will be measured in homozygous knockout-knockin ve3rsus wild-type mice. The rate of growth will also be assessed in these two groups of animals. Aim 2 will also determine if progressively higher dosages of Tet can be used to titrate the circulating levels of GH and IGF-1 in knockout-knockin mice. Initially, Tet will be administered in the drinking water. Should this approach prove to not be optimal, then subsequent studies will assess the ability of subcutaneously administered slow release tablets of Tet to titrate GH. The knockout- knockin mouse will e used in an ensuing R01 application to test the hypothesis that [1] reduced GH levels early in life may be advantageous to enhancing life span while [2] a moderate increase in GH levels in later life may also contribute to longevity.

目的是开发和初步表征一种新的转基因小鼠模型，其中GH的循环水平是正常的，但可以使用四环素抑制反式激活因子系统向下滴定。目的1是建立一种敲除-敲入转基因小鼠模型，该模型将[1]敲除内源性小鼠GH基因，[2]插入一个DNA构建体，该构建体将四环素（泰特）抑制性反式激活蛋白（tTAr）的表达置于内源性小鼠GH启动子的控制下。反式激活因子将依次激活最小的反式激活因子依赖性（TD）启动子以驱动大鼠GH基因（rGH）的表达。通过这种策略，内源性GH启动子将基本上驱动大鼠GH的表达。由于所有的正常调节机制将是完整的，GH水平应该接近正常水平，在小鼠中发现，并应显示脉冲释放这种激素的特点。目标2将通过首先确定敲除-敲入模型如何再现GH的正常分泌来表征和验证模型。将在纯合敲除-敲入ve 3rsus野生型小鼠中测量血浆GH和胰岛素样生长因子（IGF-1）。还将评估这两组动物的生长速率。目的2还将确定是否可以使用逐渐增加剂量的泰特来滴定基因敲除-基因敲入小鼠中GH和IGF-1的循环水平。最初，将在饮用水中给予泰特。如果这种方法被证明不是最佳的，则后续研究将评估皮下给药的泰特缓释片滴定GH的能力。敲除-敲入小鼠将用于随后的R 01应用中以检验以下假设：[1]生命早期GH水平降低可能有利于延长寿命，而[2]生命后期GH水平适度增加也可能有助于长寿。