Functional Analysis of Breast Cancer Susceptibility Gene

乳腺癌易感基因的功能分析

• 批准号: 6763739
• 负责人: SHYAM SHARAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6763739
• 关键词: DNA repair; artificial chromosomes; brca gene; breast neoplasms; gene mutation; genetic manipulation; genetic susceptibility; genetically modified animals; human genetic material tag; laboratory mouse; neoplasm /cancer genetics; point mutation; polymerase chain reaction; protein protein interaction; protein structure function; tumor suppressor genes; tumor suppressor proteins; yeast two hybrid system

Breast cancer is the most common disease and leading cause of lethality in women. The identification and cloning of the BRCA1 and BRCA2 genes have demonstrated that mutations in these two genes account for almost all of the families with multiple cases of breast and ovarian cancer. To understand the biological function of these genes, mutations that result in loss of function have been generated in mice. These studies have demonstrated the involvement of these two genes in cell proliferation and normal development and also in the Rad51-mediated DNA recombination repair machinery. Mutation in the Brca1 or Brca2 genes may result in defect in DNA repair/monitor system causing accumulation of mutation in growth control genes, leading to tumorigenesis. One of the goals of our research is to conduct a functional dissection of the Brca1 and Brca2 protein and to determine other components of the pathway(s) in which these function. To achieve this, we are generating subtle mutations in regions of the gene encoding these two proteins to study their phenotypic effect. To generate these multiple alleles, we are using bacterial artificial chromosomes (BACs). We have identified BAC clones that contain the functional Brca1 and Brca2 gene, as they can rescue the embryonic lethality associated with mutation in these genes. We are using BACs with mutation in these genes to generate transgenic mice on a Brca1 or Brca2 mutant background. We are manipulating the BACs by using the bacteriophage recombination system. We have developed a rapid method to manipulate BACs to generate deletions, insertions and single base changes using oligonucleotides as targeting vectors obviating the use of selection markers. We have developed a PCR-based approach to identify the correctly targeted clone. In human cancer patients, in addition to mutations that clearly disrupt the gene, several missense mutations have been identified in the BRCA1 and BRCA2 genes. The functional significance of these mutations is unknown. These may result in an unstable protein, alter a subset of its function, or represent a rare polymorphic variant. Our aim is to generate missense mutations in murine Brca1 and Brca2 genes and study their functional significance using the BAC transgenic approach. We have examined the possibility of using human gene in mice to generate missense mutations. We have generated humanized mice using human BRCA1 as well as the BRCA2 gene. These transgenic mice are able to rescue to embryonic lethality associated with mutation in the endogenous Brca1 or Brca2 genes. We are generating missense mutations in the human gene to examine their functional significance. Such mutations will serve as valuable tool for functional dissection of these genes.

乳腺癌是女性最常见的疾病，也是导致女性死亡的主要原因。 BRCA1和BRCA2基因的鉴定和克隆表明，这两个基因的突变几乎占所有乳腺癌和卵巢癌多发病例的家族。为了了解这些基因的生物学功能，在小鼠中产生了导致功能丧失的突变。这些研究证明这两个基因参与细胞增殖和正常发育，以及 Rad51 介导的 DNA 重组修复机制。 Brca1或Brca2基因突变可能导致DNA修复/监控系统缺陷，导致生长控制基因突变积累，导致肿瘤发生。 我们研究的目标之一是对 Brca1 和 Brca2 蛋白进行功能解剖，并确定这些蛋白发挥作用的途径的其他成分。为了实现这一目标，我们在编码这两种蛋白质的基因区域中产生微妙的突变，以研究它们的表型效应。为了生成这些多个等位基因，我们使用细菌人工染色体（BAC）。我们已经鉴定出含有功能性 Brca1 和 Brca2 基因的 BAC 克隆，因为它们可以挽救与这些基因突变相关的胚胎致死性。我们使用这些基因发生突变的 BAC 来生成 Brca1 或 Brca2 突变背景的转基因小鼠。我们正在通过使用噬菌体重组系统来操纵 BAC。我们开发了一种快速方法，使用寡核苷酸作为靶向载体来操纵 BAC 产生缺失、插入和单碱基变化，从而避免使用选择标记。我们开发了一种基于 PCR 的方法来识别正确的目标克隆。 在人类癌症患者中，除了明显破坏该基因的突变外，还在 BRCA1 和 BRCA2 基因中发现了一些错义突变。这些突变的功能意义尚不清楚。这些可能会导致蛋白质不稳定、改变其部分功能或代表罕见的多态性变体。我们的目标是在小鼠 Brca1 和 Brca2 基因中产生错义突变，并使用 BAC 转基因方法研究它们的功能意义。我们已经研究了在小鼠中使用人类基因产生错义突变的可能性。我们利用人类 BRCA1 和 BRCA2 基因培育出了人源化小鼠。这些转基因小鼠能够挽救与内源 Brca1 或 Brca2 基因突变相关的胚胎死亡。我们正在人类基因中产生错义突变来检查它们的功能意义。这些突变将成为这些基因功能解剖的宝贵工具。