MOLECULAR GENETICS OF THERMOGENESIS

描述：(改编自申请者摘要)：调查人员寻求 了解兼性和强制性生热作用在 对体重的调节，并找到减少 通过增加生热作用而发展成肥胖。不发抖 产热作用是棕色脂肪的主要生理功能，其基础是 线粒体解偶联蛋白的棕色脂肪特异性表达 (UCP1)，线粒体膜内的质子载体，解偶联 氧化磷酸化产生热。小鼠的遗传操作 在转基因小鼠中增加UCP1可以减少肥胖。在小鼠中， UCP1基因已被基因打靶灭活对 寒冷；然而，自相矛盾的是，他们并没有患上肥胖症。结果 表明存在替代的生热机制来补偿 UCP1的丧失。 调查人员推测，一种新发现的同源基因 UCP1，称为UCP2，是一种强制产热的机制，可以 恢复Ucp1缺陷小鼠的能量平衡。实验是 Ucp2在野生型小鼠中的表达 Ucp1基因敲除的纯合性与其在 组织间的发育，以及喂养高脂肪/高脂肪的结果 蔗糖减肥法。为了确定UCP2分子是否生热，一种 将使用双重替换技术构建转基因小鼠 在ES细胞中，将Ucp2基因插入Ucp1基因。这将是 将Ucp2基因置于交感神经系统的控制之下， 将使我们能够确定UCP2分子是否可以保护小鼠 冷暴露导致UCP1缺乏。第三个具体目标将是 用Cre-loxP噬菌体系统灭活Ucp2基因 重组。由于Ucp2是一种普遍表达的基因，因此该系统 灭活一个基因将使我们能够灭活这些组织中的Ucp2 该Cre重组酶被选择性地表达。有了这个遗传系统 对Upc2在组织能量平衡中作用的明确评估可以 才能实现。第四个具体目标是寻求产生一种新的 白喉毒素细胞特异性表达的棕色脂肪消融。 这一新模型将通过基因替换技术生成插入 将白喉毒素基因导入Ucp1基因，以实现细胞的最大化 其表达的特异性，并明确地检验了这一假设 去除棕色脂肪会导致吞噬功能亢进。

DESCRIPTION: (Adapted from applicant's abstract): The investigators seek to understand the role of facultative and obligatory thermogenesis in the regulation of body weight and to find mechanisms that will reduce the development of obesity by increasing thermogenesis. Nonshivering thermogenesis, the major physiological function of brown fat, is based on the brown fat specific expression of the mitochondrial uncoupling protein (UCP1), a proton carrier in the inner mitochondrial membrane, that uncouples oxidative phosphorylation to produce heat. Genetic manipulations in mice which increase UCP1 in transgenic mice reduce obesity. Mice in which the Ucp1 gene has been inactivated by gene targeting are extremely sensitive to cold; however, paradoxically, they do not develop obesity. The result suggests that alternative thermogenic mechanisms exist that compensate for the loss of UCP1. The investigators have hypothesized that a newly discovered homologue of Ucp1, called Ucp2, is a mechanism for obligatory thermogenesis that can restore energy balance in mice with a defective Ucp1. Experiments are proposed to characterize the expression of Ucp2 in wild-type mice in homozygous for the Ucp1 knockout with respect to its changes during development, among tissues and as a consequence of feeding high fat/high sucrose diets. To establish whether the UCP2 molecule is thermogenic, a transgenic mouse will be constructed using the double replacement technique in ES cells that will insert the Ucp2 cDNA into the Ucp1 gene. This will bring the Ucp2 gene under the control of the sympathetic nervous system and will enable us to determine whether the UCP2 molecular can protect mice deficient in UCP1 from the cold exposure. A third specific aim will inactivate the Ucp2 gene with the Cre-loxP bacteriophage system of recombination. Since Ucp2 is a ubiquitously expressed gene, this system for inactivating a gene will enable us to inactivate Ucp2 in those tissues for which Cre recombinase is selectively expressed. With this genetic system the unambiguous evaluation of the role of UPC2 in tissue energy balance can be achieved. The fourth specific aim seeks to produce a new model for the ablation of brown fat with the cell specific expression of diphtheria toxin. This new model will be generated by gene replacement techniques to insert the diphtheria toxin gene into the Ucp1 gene in order to maximize the cell specificity of its expression and test unequivocally the hypothesis that the ablation of brown fat leads to hyperphagia.