REGULATION OF GH BY A TETRACYCLINE-REPRESSIBLE SYSTEM

目的是建立并初步鉴定一种新的转基因小鼠模型，在该模型中，循环中的生长激素水平是正常的，但可以使用四环素可抑制的反式激活系统来向下滴定。目的1建立敲除转基因小鼠模型，[1]敲除内源性小鼠生长激素基因，[2]插入一个DNA构建体，将四环素(Tet)可抑制反式激活蛋白(TTAR)的表达置于内源性小鼠GH启动子的控制之下。反式激活剂将反过来激活一个最小的反式激活剂依赖(TD)启动子，以驱动大鼠生长激素基因(RGH)的表达。通过这一策略，内源性生长激素启动子将本质上驱动大鼠生长激素的表达。由于所有正常的调节机制都将完好无损，生长激素水平应该接近小鼠的正常水平，并且应该表现出这种激素的脉动性释放特征。目标2将通过首先确定敲除-敲除模型重述GH正常分泌的情况来表征和验证该模型。将测定纯合基因敲除野生型小鼠的血浆生长激素和胰岛素样生长因子(IGF-1)。这两组动物的生长速度也将得到评估。目的2还将确定是否可以使用递增剂量的Tet来滴定基因敲除小鼠循环中GH和IGF-1的水平。最初，Tet将在饮用水中使用。如果这种方法被证明不是最优的，那么后续的研究将评估皮下注射Tet缓释片滴定GH的能力。敲除基因的小鼠将被用于随后的R01应用程序，以测试[1]生命早期降低GH水平可能有利于延长寿命的假设，而[2]晚年GH水平适度增加也可能有助于长寿。

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.