Inducible LIF Receptor Ablation in Adult Mice

在成年小鼠中选择性地、诱导地和可逆地针对特定蛋白质的突变，这种能力将成为研究衰老的有力工具。为了实现这一目标，我们对ES细胞中的白血病抑制因子受体（LIFR）基因进行了四环素反应性消融。这种突变通过小鼠生殖系传播，针对目标突变的杂合交配对现在正在产生幼崽。使用标准的非诱导基因靶向技术导致的LIFR缺失是一种围产期致死性疾病，会深刻影响许多系统，包括骨（骨质疏松症）和胶质细胞发育（胶质细胞生成）。因此，我们现在准备解决：四环素诱导基因消融方法在衰老研究中的应用确定成年后LIFR缺失的后果。该诱导型靶向载体包含一套完整的触发元件，从而将全长的大鼠LIFR cDNA同源整合到小鼠LIFR的外显子2中，有效地将小鼠基因与四环素控制引入的大鼠LIFR同源物相结合。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的表达是在小鼠LIFR在缺乏四环素衍生物强力霉素（Dox）的情况下组成性表达的地方，并且在Dox存在时沉默。去除Dox后，大鼠LIFR的表达被重新激活。该系统的敏感性将通过半定量反转录聚合酶链反应（rtPCR）来研究，以测量饮用水中阿霉素对整个组织LIFR水平的影响，并利用靶向构建中的β -半乳糖苷酶报告基因来研究，该基因在阿霉素存在时也会关闭。β -半乳糖苷酶将通过X-gal染色原位可见，以分析Dox给药的局部效应。Dox的影响和成人LIFR消融生物学后果的初步分析将在骨骼、中枢神经系统、骨骼肌和心肌、肺、肝、胰腺、脾和肾中进行评估。总之，一种新的成人基因操作技术将被开发和表征，这将阐明利用LIFR的多功能细胞因子在衰老中的作用。

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.