Inducible LIF Receptor Ablation in Adult Mice

在成年小鼠中选择性地、可诱导地和可逆地针对特定蛋白质进行突变的能力将是研究衰老的有力工具。为了达到这个目标，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。这种突变已经通过小鼠的生殖系传播，目标突变的杂合性交配对现在正在生产幼崽。使用标准的非诱导基因打靶技术导致的LIFR丢失是一种围产期的致命性疾病，严重影响许多系统，包括骨质疏松和神经胶质细胞发育(无分化)。因此，我们现在准备解决：1.四环素诱导基因消融方法在衰老研究中的应用2.鉴定成人LIFR缺失的后果。该可诱导的靶向载体整合了一套完整的tet-off元件，使得全长的大鼠LIFR基因被同源地整合到小鼠LIFR的第二外显子中，有效地通过四环素控制引入的大鼠LIFR同源物来消融小鼠基因。由于大鼠LIFR插入的靶点是在内源性小鼠LIFR启动子元件的适当控制下，因此大鼠LIFR的表达是在缺乏四环素衍生物多西环素(Dox)的情况下结构性表达的，在Dox存在的情况下是沉默的。去除Dox后，大鼠LIFR的表达重新激活。该系统的敏感性将通过半定量逆转录聚合酶链式反应(RTPCR)来测量饮用水中Dox对整个组织LIFR水平的变化，并通过利用靶向构建体中掺入的β-半乳糖苷酶报告基因来研究，该基因在Dox存在时也被关闭。β-半乳糖苷酶将通过X-Gal染色进行原位观察，以分析Dox给药的局部效应。Dox的效果和成人LIFR消融的生物学后果的初步分析将在骨骼、中枢神经系统、骨骼和心肌、肺、肝、胰腺、脾和肾脏进行评估。总之，将开发一种新的成人基因操作技术，并对其进行表征，以阐明利用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.