Identification of genetic interactors of Brca2

Brca2 遗传相互作用子的鉴定

基本信息

批准号：
10486808
负责人：
SHYAM SHARAN
金额：
$ 106.42万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10486808
关键词：
Address Affect Alleles BRCA deficient BRCA1 gene BRCA2 gene Biological Breast Candidate Disease Gene Cell Cycle Arrest Cell Cycle Checkpoint Cell Cycle Checkpoint Genes Cell Death Cell division Cells Cloning DNA DNA Damage DNA Repair ES Cell Line Embryo Epithelial Financial compensation Gene Expression Genes Genetic Genetic Recombination Genetic Screening Germ-Line Mutation Goals Growth Human Hypersensitivity Insertional Mutagenesis Lead Libraries Lower Organism Malignant neoplasm of ovary Mediating Mus Mutation Neoplasms Ovarian Pathway interactions Phenotype Process Proliferating Retroviral Vector Retroviridae Role Site Small Interfering RNA Somatic Cell Testing Therapeutic Intervention Tissues Viral Virus base cell type coping embryonic stem cell high throughput screening homologous recombination inhibitor/antagonist loss of function malignant breast neoplasm mutant neoplastic cell repaired response stem cells targeted treatment tumor tumorigenesis

项目摘要

We will perform insertional mutagenesis using murine stem cell virus (MSCV) to activate genes in ES cells using the viral LTR. The advantage of using retroviruses for insertional mutagenesis is that cloning of the insertion sites is relatively simple. ES cells with randomly inserted MSCV will be selected for viable clones after both copies of the functional Brca1 or Brca2 are disrupted. The advantage of using MSCV is that, unlike other retroviral vectors, its LTR can drive high levels of target gene expression in ES cells. In a complementary approach, we will also use an siRNA library-based screen to identify genes whose loss of function can rescue the lethality of BRCA2-deficient ES cells. We will take advantage of the intact DNA damage response machinery and cell-cycle checkpoint genes of ES cells to perform an siRNA-based screen to identify genes whose loss can rescue the lethality of Brca2 null ES cells. Identification of such genes will provide clues to the genes that allow BRCA2-deficient somatic cells to survive and develop into tumors. To allow rapid high-throughput screening, we have generated a mouse ES cell line that expresses a mutant allele of BRCA2 that is defective in homologous recombination and renders the cells hypersensitive to PARP inhibitors. In the primary screen we will identify siRNAs that allow these ES cells to survive in the presence of PARP inhibitors. The results obtained will provide clues to the genes may compensate for the loss of BRCA2-mediated homologous recombination. To further address the specific compensation for the loss of function of BRCA2, we will perform a secondary screen, in which we will test the siRNAs for their ability to rescue the lethality of Brca2-null ES cells. For this screen, we have generated a line of mouse ES cells that have one functionally null allele of Brca2 and the second allele is conditional and flanked by two loxP sites. The conditional allele can be deleted by Cre-mediated recombination. We will examine the expression of candidate genes in human BRCA2-deficient tumors to identify those that are silenced or down regulated. We believe that genes that contribute to BRCA2-mediated tumorigenesis can be potential targets for therapeutic intervention if their expression in tumor cells can lead to cell death or cell cycle arrest.

我们将使用鼠干细胞病毒 (MSCV) 进行插入诱变，以利用病毒 LTR 激活 ES 细胞中的基因。使用逆转录病毒进行插入诱变的优点是插入位点的克隆相对简单。在功能性 Brca1 或 Brca2 的两个拷贝都被破坏后，随机插入 MSCV 的 ES 细胞将被选择用于存活克隆。使用MSCV的优点在于，与其他逆转录病毒载体不同，其LTR可以驱动ES细胞中靶基因的高水平表达。在一种补充方法中，我们还将使用基于 siRNA 库的筛选来识别功能丧失的基因，这些基因可以挽救 BRCA2 缺陷型 ES 细胞的杀伤力。我们将利用 ES 细胞完整的 DNA 损伤反应机制和细胞周期检查点基因进行基于 siRNA 的筛选，以鉴定其丢失可以挽救 Brca2 null ES 细胞致死性的基因。这些基因的鉴定将为BRCA2缺陷体细胞存活并发展成肿瘤的基因提供线索。为了进行快速高通量筛选，我们生成了表达 BRCA2 突变等位基因的小鼠 ES 细胞系，该突变等位基因在同源重组方面存在缺陷，并使细胞对 PARP 抑制剂过敏。在初级筛选中，我们将鉴定允许这些 ES 细胞在 PARP 抑制剂存在下存活的 siRNA。获得的结果将为基因可能补偿 BRCA2 介导的同源重组的损失提供线索。为了进一步解决 BRCA2 功能丧失的具体补偿问题，我们将进行二次筛选，其中我们将测试 siRNA 拯救 Brca2-null ES 细胞致死性的能力。对于此筛选，我们生成了一系列小鼠 ES 细胞，其具有一个 Brca2 功能无效等位基因，第二个等位基因是条件等位基因，且两侧有两个 loxP 位点。条件等位基因可以通过 Cre 介导的重组来删除。我们将检查人类 BRCA2 缺陷肿瘤中候选基因的表达，以确定那些被沉默或下调的基因。我们相信，如果有助于 BRCA2 介导的肿瘤发生的基因在肿瘤细胞中的表达能够导致细胞死亡或细胞周期停滞，那么它们可以成为治疗干预的潜在靶点。