Generation of ES Cells to Tissue-Specifically Overexpress Nuclear Receptors and C

通过去除小鼠的重要基因而导致的功能丧失通常被用于 建立动物模型以研究受体和受体在疾病中的作用。然而，许多人 疾病是由通过扩增或过度表达辅调节因子和 感受器。因此，在小鼠体内以组织特异性的方式过度表达这些基因无疑将有助于我们 了解这些受体的生理功能。在这个项目中，我们建议生成ES细胞 可以用来过度表达所有49个小鼠核受体和与代谢相关的&gt；10辅助调节因子 疾病。协同调节器将包括：PGC1a、SRC-3、SRC-2、SRC-1、RIP140和其他显示的协同调节器 对新陈代谢和心血管疾病、癌症和衰老很重要。这些ES细胞可以：(1)使用 使小鼠在任何组织中以组织特异性的方式过度表达这些因子 重组酶小鼠系可用于或(2)研究其过表达对干细胞的影响 功能。由于重组频率非常高，ES细胞将以高吞吐量的方式产生 在罗莎轨迹的高处。此外，我们将从这些ES细胞中产生有限数量的成年小鼠，用于 原则性证明。 为了产生敲门过表达的ES细胞，我们将利用一种独特的程序。我们已经生成了一个 用CAGGS启动子引导核受体和辅助调节因子的ROSA基因座靶向载体 在CAGGS启动子和启动子之间插入了loxP-Stop-loxP盒 辅调节因子/受体编码序列，防止表达，直到重组发生。当老鼠窝藏在 这种敲击信号被交叉到组织特异性CRE小鼠身上，协同调节/受体基因将被激活，并 在表达Cre的任何组织中表达。该系统具有许多优点：(1)不表达 在缺乏Cre重组的小鼠中发现感兴趣的基因；(2)单个小鼠品系可以用于过度表达 在任何给定的感兴趣组织中的协同调节/受体；(3)在以下情况下可以影响时间表达 使用Cre的诱导表达(他莫昔芬或四环素诱导型Cre)；(4)确保均一 在任何感兴趣的组织中的表达水平；(5)该位点/启动子将插入的基因的表达限制为NO 比内生水平高出几倍以上；(6)在类似的标志下，调控的显性负面 分子可以在特定的组织中表达。 一旦胚胎干细胞产生，冷冻的胚胎干细胞将可供该领域的所有研究人员使用。 同样，我们培育的小鼠产生的胚胎也将免费分发给研究人员 请求。此外，还将向任何领域的调查人员提供易于使用的靶向盒。

Loss of function through ablation of genes of importance in mice has been commonly employed in the field to generate animal model to study receptor and cpregulator function in diseases. However, many diseases are caused by gain of function through amplification or over-expression of coregulators and receptors. Thus, over expression of these genes in mice in a tissue specific manner will undoubtedly aid our understanding of the physiological function of these receptors. In this project, we propose to generate ES cells that can be used to over-express all 49 murine nuclear receptors and >10 coregulators relevant to metabolic diseases. The coregulators will include: PGC1a, SRC-3, SRC-2, SRC-1, RIP140 and other coregulators shown to be important for metabolic and cardiovascular diseases, cancer and aging. These ES cells can be: (1) used to produce mice over-expressing these factors in a tissue-specific manner in any tissue where specific Cre recombinase mouse line is available or (2) used to investigate the effect of their over-expression on stem cell function. The ES cells will be generated in a 'high throughput' fashion, since recombination frequency is very high in the ROSA locus. In addition, we will generate a limited number of adult mice from these ES cells for proof of principle. To generate knockin over-expressing ES cells, we will utilize a unique procedure. We have generated a targeting vector for the ROSA locus with a CAGGS promoter directing nuclear receptors and coregulators expression; a LoxP-Stop-LoxP cassette is inserted in between the CAGGS promoter and the coregulator/receptor coding sequence to prevent expression until recombination occurs. When mice harboring this knockin are crossed to tissue-specific Cre mice, the coregulator/receptor gene will be turned on and expressed in any tissue where the Cre is expressed. This system has many advantages: (1) no expression of gene of interest in mice in the absence of the Cre recombination; (2) a single mouse line can be used to overexpress coregulator/receptors in any given tissue of interest; (3) temporal expression can be effected if inducible expression of Cre is employed (tamoxifen or tetracycline inducible Cre); (4) ensure uniform expression level in any tissue of interest; (5) the locus/promoter limits expression of the inserted gene to no more than a few fold over the endogenous level; (6) in a similar token dominant negatives of regulatory molecules can be expressed in specific tissues. Once ES cells are generated, frozen ES cells will be made available to all investigators in the field. Similarly, embryos generated from the mice we produced will also be freely distributed to investigator upon request. In addition, easily used targeting cassettes will be made available for investigators in any field.