Single and Double Knockout of TR2 and TR4 Receptors

描述（由申请人提供）：我们提出的研究目标是探索核孤儿受体TR2和TR4的靶向破坏的影响，以及两个基因组合破坏的后果，以确定这些受体的生理作用。我们假设TR2和TR4都在发育、生理和行为系统的调节中发挥重要作用。为了确定TR2和TR4在体内的具体作用，我们将采用显示任一孤儿受体消融的小鼠模型，以及同时显示两种受体消融的动物模型。特异性目的1:TR4敲除/ β -gal敲除蛋白（TR4 -/-）小鼠的表征。特异性目的2:TR2敲除jbeta -gal敲除蛋白（TR2 -/-）小鼠的表征。我们假设这种受体在早期发育阶段更为重要，并且TR2 -/-小鼠可能在出生前或出生后死亡。将通过形态学和组织学分析调查死亡率，并评估存活至成年的动物的生长速度和生育力。特异性目的3:TR2/TR4双敲除小鼠的表征。双敲除动物的分析将按照Specific Aim 2中描述的方法进行，并将表型与TR2敲除、TR4敲除和野生型小鼠进行比较分析。专项目标4：分析TR4和/或TR2消融对靶基因调控的影响。TR4、TR2和TR2/TR4敲除动物将成为研究这些孤儿受体在体内系统中已知靶基因的工具，并证实所鉴定的调控通路的生理意义，为筛选新的TR2/TR4靶基因提供材料来源。4a：测定TR4和/或TR2靶基因消融对已知靶基因表达的影响。比较敲除动物与野生型对照中TR4和TR2下游靶点的内源性基因表达和蛋白水平。4b：通过基因微阵列技术鉴定TR4和/或TR2独特或差异调控的新基因，我们希望剖析TR4和TR2介导靶基因调控的差异，从而进一步了解这些神秘的孤儿受体在哺乳动物发育和生理中的作用。综上所述，这一提议为我们研究TR2和TR4孤儿核受体在体内的潜在生理作用提供了第一次机会。

DESCRIPTION (provided by applicant): The goal of our proposed research is to explore the effects of targeted disruption of the nuclear orphan receptors TR2 and TR4, as well as consequences of disruption of the two genes in combination, in order to determine the physiological roles of these receptors. We hypothesize that both TR2 and TR4 play significant roles in the regulation of developmental, physiological, and behavioral systems. To determine the specific roles of TR2 and TR4 in vivo, we will employ mouse models exhibiting ablation of either orphan receptor, as well as animals exhibiting simultaneous ablation of both receptors. Specific aim 1: Characterization of TR4 knockout/Beta-gal knockin (TR4 -/-) mice. Specific aim 2: Characterization of TR2 knockoutJBeta-gal knockin (TR2 -/-) mice. We hypothesize this receptor is more important in early stages of development, and that TR2 -/- mice may die pre- or postnatally. Mortality will be investigated by morphological and histological analyses, and animals surviving to adulthood will be assessed for growth rate and fertility. Specific aim 3: Characterization of TR2/TR4 double knockout mice. Analysis of the double knockout animals will follow the approach described in Specific Aim 2, and the phenotypes will be analyzed in comparison to TR2 knockout, TR4 knockout, and wildtype mice. Specific Aim 4: Analysis of the effects of TR4 and/or TR2 ablation on target gene regulation. TR4, TR2, and TR2/TR4 knockout animals will be tools to study the known target genes of these orphan receptors in an in vivo system, and to confirm the physiological significance of the identified regulatory pathways, and provide sources of material for the screening of novel TR2/TR4 target genes. 4a: Determination of effect of TR4 and/or TR2 target gene ablation on known target gene expression. Compare endogenous gene expression, and protein levels of TR4 and TR2 downstream targets in knockout animals versus wildtype controls. 4b: Identifiy novel genes regulated uniquely or differentially by TR4 and/or TR2 through the use of gene microarray technology, we hope to dissect the differences in target gene regulation mediated by TR4 and TR2 and, in this way, further understand the roles of these enigmatic orphan receptors in mammalian development and physiology. In summary, this proposal provides us the first opportunity to study the potential in vivo physiological roles of the TR2 and TR4 orphan nuclear receptors.