Inducible LIF Receptor Ablation in Adult Mice

成年小鼠中诱导型 LIF 受体消融

• 批准号: 6331979
• 负责人: CAROL B WARE
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331979
• 关键词: RNA; aging; animal genetic material tag; beta galactosidase; biotechnology; cytokine receptors; gene induction /repression; gene targeting; genetic promoter element; genetically modified animals; histology; homeostasis; laboratory mouse; leukemia inhibitory factor; mature animal; polymerase chain reaction; technology /technique development; tetracyclines; tissue /cell preparation; transfection

The ability to selectively, inducibly and reversibly target mutations to specific proteins in adult mice would be a powerful tool in the study of aging. Toward this goal, we have made a tetracycline responsive ablation of the gene for the leukemia inhibitory factor receptor (LIFR) in ES cells. This mutation has transmitted through the mouse germline and mating pairs heterozygous for the targeted mutation are now producing pups. Loss of LIFR using standard non-inducible gene targeting techniques is a perinatal lethal profoundly affecting many systems including bone (osteoporosis) and glial cell development (agliogenesis). Thus, we are now poised to address: 1. the utility of a tetracycline inducible gene ablation approach in the study of aging 2. identification of adult consequences of LIFR loss. The inducible targeting vector incorporates a complete set of tet-off elements so that a full length rat LIFR cDNA is incorporated homologously into exon 2 of the mouse LIFR effectively ablating the mouse gene with tetracycline control of the introduced rat LIFR homolog. Because rat LIFR insertion is targeted to be under appropriate control of the endogenous mouse LIFR promoter elements, expression of rat LIFR is on where murine LIFR is constitutively expressed in the absence of a tetracycline derivative, doxycycline (Dox) and silenced in the presence of Dox. Expression of rat LIFR is reactivated upon removal of Dox. Sensitivity of this system will be studied both by semi- quantitative reverse transcription polymerase chain reaction (rtPCR) to measure whole tissue alterations in LIFR levels in response to Dox in the drinking water and by utilizing a beta-galactosidase reporter gene incorporated in the targeting construct which is also switched off in the presence of Dox. Beta-galactosidase will be visualized in situ by X-gal staining to analyze localized effects of Dox administration. Effects of Dox and preliminary analysis of biological consequences of adult LIFR ablation will be assessed in bone, the central nervous system, skeletal and cardiac muscle, lung, liver, pancreas, spleen and kidney. In summary, a new technique for adult genetic manipulation will be developed and characterized that will elucidate the role in aging of the multi-functional cytokines that utilize LIFR.

在成年小鼠中选择性，诱导和可逆性地靶向突变的能力将是衰老研究的强大工具。为了实现这一目标，我们已经对ES细胞中白血病抑制因子受体（LIFR）的基因进行了四环素反应性消融。该突变通过小鼠种系传输，并为靶向突变的杂合子配对杂合子正在产生幼犬。使用标准的不可诱导基因靶向技术损失LIFR是一种围产期致死性，对许多系统（包括骨骼（骨质疏松症）和神经胶质细胞发育（Agliogenesen））的许多系统产生了深远影响。因此，我们现在准备解决：1。在衰老2研究中，四环素诱导基因消融方法的实用性。确定LIFR损失的成年后果。诱导的靶向矢量结合了一组Tet-Off元件集，因此将全长大鼠LIFR cDNA同源地纳入了小鼠LIFR的外显子2，从而通过引入大鼠LIFR同源物的四环素控制有效地烧毁了小鼠基因。由于大鼠LIFR插入的目标是在内源性小鼠LIFR启动子元件的适当控制下，因此大鼠LIFR的表达在没有四环素衍生物，多克环素（DOX）的情况下组成型鼠LIFR的表达，并在DOX的情况下进行沉默。去除DOX后，大鼠LIFR的表达被重新激活。将通过半定量逆转录聚合酶链反应（RTPCR）研究该系统的敏感性，以测量LIFR水平的整个组织变化，以响应饮用水中的DOX，并利用在靶向结构中掺入的β-半乳糖苷酶测试剂基因，该基因还掺入了DOX的存在。 β-半乳糖苷酶将通过X-GAL染色在原位可视化，以分析DOX给药的局部作用。 DOX和对成人LIFR消融生物学后果的初步分析将在骨，中枢神经系统，骨骼和心脏肌肉，肺，肝，胰腺，胰腺，脾和肾脏中评估。总而言之，将开发和表征一种针对成人遗传操作的新技术，该技术将阐明利用LIFR的多功能细胞因子衰老中的作用。