Conditional Protein Targeting

描述(由申请人提供)：人类疾病的动物模型极大地促进了医学进步。近年来，通过使用同源重组来干扰小鼠的特定基因，哺乳动物发育和生理学的研究已经发生了革命性的变化。然而，对具有零突变的基因敲除小鼠的表型的解释往往受到早期胚胎死亡以及发育过程中缺乏基因的细胞和分子补偿的影响。为了缓解这些缺点，已经开发了允许有条件地使基因失活的方法，但这些方法通常是缓慢的和不可逆转的。一种更可取的方法是可逆地针对特定基因的蛋白质产物，而不是基因本身。这项研究的广泛和长期目标是开发新的策略，允许在细胞培养和动物中有条件地靶向特定的蛋白质。已经开发出一种实验系统，其中特定蛋白质的功能取决于合成的、细胞可渗透的有机分子的存在或不存在。这种合成的分子与一个小的蛋白结构域紧密结合，该蛋白结构域通过同源重组与感兴趣的蛋白质融合，创造出表达嵌合蛋白的敲入小鼠。在没有合成分子的情况下，嵌合蛋白被结构性地灭活。在加入细胞培养液或给小鼠注射后，合成的分子与嵌合蛋白中的受体结合，并恢复其功能。合成分子的退出逆转了这一过程，并导致嵌合蛋白迅速失活。这项新技术可以在细胞培养或小鼠体内对特定蛋白质进行快速和可逆的调节。拟议研究的具体目标集中在合成具有更好药理学特性的新的蛋白质配体，以及鉴定与这些合成衍生物紧密和特异结合的新的蛋白质受体。这种有条件的蛋白质靶向策略也将被用于探索特定蛋白质在不同时间点在小鼠发育中的作用。

DESCRIPTION (provided by applicant): Medical advances are aided enormously by animal models of human diseases. In recent years, studies of mammalian development and physiology have been revolutionized through the use of homologous recombination to disrupt specific genes in mice. However, interpretation of the phenotypes of knockout mice possessing null mutations is often clouded by early embryonic lethality as well as cellular and molecular compensation for the absence of a gene during development. To mitigate these shortcomings, methods that allow conditional inactivation of genes have been developed, but these methods are generally slow and irreversible. A more desirable approach would be to reversibly target the protein product of a specific gene rather than the gene itself. The broad, long-term objectives of this research are to develop new strategies that allow conditional targeting of specific proteins in cell culture and in animals. An experimental system has been developed in which the function of a specific protein of interest depends on the presence or absence of a synthetic, cell permeable organic molecule. This synthetic molecule binds tightly to a small protein domain that is fused to a protein of interest using homologous recombination to create knock-in mice that express the chimeric protein. In the absence of the synthetic molecule, the chimeric protein is constitutively inactivated. Upon addition to cell culture media or administration to a mouse, the synthetic molecule binds to its receptor within the chimeric protein and restores its function. Withdrawal of the synthetic molecule reverses this process and causes the chimeric protein to be rapidly inactivated. This new technique allows rapid and reversible regulation of a specific protein either in cell culture or in mice. The specific aims of the proposed research focus on the synthesis of new protein ligands that possess better pharmacological properties as well as the identification of new protein receptors that bind tightly and specifically to these synthetic derivatives. This conditional protein targeting strategy will also be used to probe the roles of specific proteins at different time points in mouse development.