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

产生组织特异性过表达核受体和 C 的 ES 细胞

• 批准号: 7350624
• 负责人: Ming-Jer Tsai
• 金额: $ 8.87万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7350624
• 关键词: Ablation; Adult; Aging; Animal Model; Cardiovascular Diseases; Cell physiology; Code; Communities; Disease; Dominant-Negative Mutation; Embryo; Ensure; Facility Construction Funding Category; Freezing; Frequencies; Gene Expression; Generations; Genes; Genetic Recombination; Internet; Knock-in Mouse; Malignant Neoplasms; Metabolic; Metabolic Diseases; Mus; NRIP1 gene; Nuclear Receptors; Numbers; Physiological; Procedures; Protein Overexpression; Receptor Gene; Research Personnel; Resources; Stem cells; System; Tamoxifen; Tetracycline; Tetracyclines; Tissues; embryonic stem cell; expression vector; gain of function; homologous recombination; interest; loss of function; mouse Cre recombinase; nuclear receptor coactivator 1; prevent; promoter; receptor; receptor function; vector

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.

通过消除小鼠重要基因而丧失功能已被普遍用于 领域生成动物模型来研究疾病中的受体和 cpregulator 功能。然而，许多 疾病是由于辅助调节因子的扩增或过度表达而获得功能而引起的 受体。因此，这些基因在小鼠中以组织特异性方式过度表达无疑将有助于我们 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 可用于过表达所有 49 种小鼠核受体和 >10 种与代谢相关的辅助调节因子 疾病。核心调节器将包括：PGC1a、SRC-3、SRC-2、SRC-1、RIP140 和所示的其他核心调节器 对代谢和心血管疾病、癌症和衰老很重要。这些 ES 细胞可以： (1) 使用 产生在任何含有特定 Cre 的组织中以组织特异性方式过度表达这些因子的小鼠 重组酶小鼠系可用或 (2) 用于研究其过度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。 ROSA 基因座高。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 原理证明。 为了产生敲入过度表达的 ES 细胞，我们将采用独特的程序。我们已经生成了一个 ROSA 基因座的靶向载体，带有指导核受体和共调节子的 CAGGS 启动子 表达; LoxP-Stop-LoxP 盒插入到 CAGGS 启动子和 核心调节子/受体编码序列，以防止表达直至重组发生。当老鼠窝藏时 将这种敲入与组织特异性 Cre 小鼠杂交，核心调节器/受体基因将被打开并且 在表达 Cre 的任何组织中表达。该系统具有很多优点：（1）无需表达 在没有 Cre 重组的情况下，小鼠中感兴趣的基因； (2) 单鼠线可用于过表达 任何给定感兴趣组织中的核心调节器/受体； (3) 时间表达可以被影响，如果 使用Cre的诱导型表达（他莫昔芬或四环素诱导型Cre）； (4)保证统一 任何感兴趣组织中的表达水平； (5) 基因座/启动子将插入基因的表达限制为无 比内源水平高出几倍以上； (6) 类似代币监管的显性负面影响 分子可以在特定组织中表达。 ES 细胞生成后，冷冻 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 要求。此外，易于使用的靶向盒将为任何领域的研究人员提供。