Role of Mre11 in Lymphocyte Development and DNA Repair

本项目的目的是研究小鼠Mre11在淋巴细胞发育和一般DNA修复中的作用。酵母Mre11是被称为非同源末端连接(NHEJ)的途径的成员，该途径通过DNA双链断裂来修复染色体。在哺乳动物中，该途径的其他成员对于V(D)J重组和类开关重组是必不可少的，这两种重组产生了由淋巴细胞产生的各种抗体和T细胞受体。在人类中，这些过程中的缺陷会导致免疫缺陷和恶性肿瘤。Mre11在进化上是保守的，已知是一种内切/外切核酸酶，它至少与另外两种蛋白质Rad50和NBS形成复合体。不存在缺乏mre11的小鼠模型，这阻碍了在哺乳动物中研究这种蛋白。本文提出了这样一种鼠标的构建方案。该项目有以下具体目标：(1)产生小鼠Mre11等位基因，这些等位基因可以削弱生化活性，但保持形成蛋白质复合体的能力。(2)将这些等位基因整合到小鼠胚胎干细胞基因组中。(3)从这些干细胞中产生小鼠。这项工作所产生的细胞和小鼠将使我们能够确定Mre11‘S在VDJ重组、类别转换和一般DNA修复中的作用。本申请是为一位接受过分子生物学研究生培训和病理学住院医师培训的申请者颁发临床科学家发展导师奖(K08)。候选人的长期目标是建立和指导一个独立的研究实验室，研究免疫系统和DNA修复的基础和临床方面。虽然在微生物遗传学和重组DNA方面经验丰富，但候选人需要获得这一发展奖，以获得免疫学和将老鼠用作模型系统方面的经验。这项基本培训将在哈佛医学院血液研究中心的Frederick Alt博士的赞助下进行。

The goal of this project is to investigate the roles of mouse Mre11 in lymphocyte development and general DNA repair. Yeast Mre11 is a member of a pathway called non-homologous end joining (NHEJ), which repairs chromosomes with DNA double strand breaks. In mammals, other members of this pathway are essential for V(D)J recombination and class switch recombination, which generate the diverse repertoire of antibodies and T cell receptors produced by lymphocytes. In Humans, defects in these processes result in immunodeficiency and malignancy. Mre11 is evolutionarily conserved, and is known to be an endo/exonuclease which forms a complex with at least two other protein, Rad50 and NBS. No mouse model deficient in Mre11 exists, hindering the study of this protein in mammals. The construction of such a mouse is proposed here. This project entails the following specific aims: (1) To generate mouse mre11 alleles which cripple biochemical activity but maintain the ability to form protein complexes. (2) To engineer these alleles into the genome of mouse embryonic stem cells. (3) To generate mice from these stem cells. The cells and mice resulting from this work will allow us to determine Mre11's role in VDJ recombination, class switching, and general DNA repair. This application is for a Mentored Clinical Scientist Development Award (K08) to an applicant who has received graduate training in molecular biology, and residency training in Pathology. The candidate's long term goals are to establish and direct an independent research laboratory studying the basic and clinical aspects of the immune system and DNA repair. While experienced in microbial genetics and recombinant DNA, the candidate requires this development award to gain experience in immunology and the use of mouse as a model system. This essential training will be conducted under the sponsorship of Dr. Frederick Alt in the Center for Blood Research at the Harvard Medical School.