Single and Double Knockout of TR2 and TR4 Receptors

描述（由申请人提供）：我们拟议研究的目标是探索靶向破坏核孤儿受体TR 2和TR 4的影响，以及两种基因联合破坏的后果，以确定这些受体的生理作用。我们推测TR 2和TR 4在发育、生理和行为系统的调节中起重要作用。为了确定TR 2和TR 4在体内的具体作用，我们将采用表现出任一孤儿受体消融的小鼠模型，以及表现出两种受体同时消融的动物。具体目的1：TR 4敲除/β-gal敲入（TR 4-/-）小鼠的表征。具体目的2：TR 2敲除/β-gal敲入（TR 2-/-）小鼠的表征。我们假设这种受体在发育的早期阶段更重要，TR 2-/-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研究死亡率，并评估存活至成年期的动物的生长速率和生育力。具体目标3：TR 2/TR 4双敲除小鼠的表征。双敲除动物的分析将遵循特定目标2中描述的方法，并将与TR 2敲除、TR 4敲除和野生型小鼠相比分析表型。具体目的4：分析TR 4和/或TR 2消融对靶基因调控的影响。TR 4、TR 2和TR 2/TR 4敲除动物将成为在体内系统中研究这些孤儿受体的已知靶基因的工具，并证实所鉴定的调控通路的生理意义，并为筛选新的TR 2/TR 4靶基因提供材料来源。4a：确定TR 4和/或TR 2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TR 4和TR 2下游靶标的内源性基因表达和蛋白水平。图4 b：通过基因微阵列技术鉴定TR 4和/或TR 2独特或差异调控的新基因，我们希望能够剖析TR 4和TR 2介导的靶基因调控的差异，从而进一步了解这些神秘孤儿受体在哺乳动物发育和生理中的作用。总之，这一建议为我们提供了第一次机会，研究潜在的体内生理作用的TR 2和TR 4孤儿核受体。

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.