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

通过切除小鼠中的重要基因而导致的功能丧失已被普遍应用于治疗中。 领域产生动物模型来研究受体和cregulator在疾病中的功能。但不少 疾病是由通过辅助调节因子的扩增或过表达而获得的功能引起的， 受体。因此，这些基因在小鼠中以组织特异性方式的过度表达无疑将有助于我们的研究。 了解这些受体的生理功能。在这个项目中，我们建议产生ES细胞， 其可用于过表达所有49种鼠核受体和>10种与代谢相关的辅调节因子， 疾病共调节子将包括：PGC 1a、SRC-3、SRC-2、SRC-1、RIP 140和所示的其它共调节子 对代谢和心血管疾病、癌症和衰老都很重要。这些ES细胞可以：（1）用于 以组织特异性的方式在任何组织中过度表达这些因子， 重组酶小鼠系可用或（2）用于研究它们的过表达对干细胞的影响 功能ES细胞将以“高通量”方式产生，因为重组频率非常高。 在ROSA基因座中的高水平。此外，我们将从这些ES细胞中产生有限数量的成年小鼠， 原则的证明。 为了产生敲入过表达ES细胞，我们将利用独特的程序。我们创造了一个 具有指导核受体和辅助调节子的CAGGS启动子的ROSA基因座的靶向载体 将LoxP-Stop-LoxP盒插入CAGGS启动子和启动子之间。 辅调节子/受体编码序列，以防止表达，直到重组发生。当老鼠窝藏 这种敲入与组织特异性Cre小鼠杂交，辅调节因子/受体基因将被打开， 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 在不存在Cre重组的情况下，在小鼠中过表达目的基因;（2）可以使用单个小鼠系来过表达目的基因， 在任何给定的感兴趣的组织中的共调节子/受体;（3）如果 使用Cre的诱导型表达（他莫昔芬或四环素诱导型Cre）;（4）确保均匀的 （5）基因座/启动子将插入基因的表达限制为不表达， 超过内源性水平的几倍;（6）在类似的象征性显性负调控中， 分子可以在特定组织中表达。 一旦ES细胞产生，冷冻的ES细胞将提供给该领域的所有研究人员。 同样，我们生产的小鼠产生的胚胎也将免费分发给研究人员， 请求过程中此外，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.