Inducible LIF Receptor Ablation in Adult Mice

在成年小鼠中选择性地、诱导性地和可逆地将突变靶向特定蛋白质的能力将是衰老研究中的有力工具。为了实现这一目标，我们在ES细胞中对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生殖系传播，并且针对靶向突变的杂合交配对现在正在产生幼崽。使用标准的非诱导型基因靶向技术的LIFR的丧失是围产期致死性的，其深刻地影响许多系统，包括骨（骨质疏松症）和神经胶质细胞发育（胶质细胞生成）。因此，我们现在准备解决：1。四环素诱导基因切除方法在衰老研究中的应用2.识别LIFR丧失的成人后果。诱导型靶向载体掺入了一整套tet-off元件，使得全长大鼠LIFR cDNA同源地掺入小鼠LIFR的外显子2中，有效地消除了引入的大鼠LIFR同系物的四环素控制的小鼠基因。因为大鼠LIFR插入被靶向在内源性小鼠LIFR启动子元件的适当控制下，大鼠LIFR的表达在鼠LIFR在不存在四环素衍生物、多西环素（Dox）的情况下组成型表达并且在存在Dox的情况下沉默的地方。大鼠LIFR的表达在去除Dox后被重新激活。将通过半定量逆转录聚合酶链反应（rtPCR）来研究该系统的灵敏度，以测量响应于饮用水中的Dox的LIFR水平的全组织变化，并通过利用掺入靶向构建体中的β-半乳糖苷酶报告基因来研究该系统的灵敏度，该靶向构建体在Dox存在下也被关闭。通过X-gal染色原位观察β-半乳糖苷酶，以分析Dox给药的局部效应。将在骨骼、中枢神经系统、骨骼肌和心肌、肺、肝、胰腺、脾和肾中评估Dox的作用和成人LIFR消融的生物学后果的初步分析。总之，一种新的成人遗传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.