MOUSE MODEL FOR HUMAN LOSS OF HETEROZYGOSITY

人类杂合性缺失的小鼠模型

• 批准号: 6203490
• 负责人: JAY Arnold TISCHFIELD
• 金额: $ 13.47万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6203490
• 关键词: DNA damage; adenine phosphoribosyltransferase; alkylating agents; alleles; cell type; chemical carcinogen; cytogenetics; embryonic stem cell; environmental toxicology; fibroblasts; fluorescent in situ hybridization; gene frequency; gene mutation; gene targeting; genetic markers; genotype; ionizing radiation; kidney cell; loss of heterozygosity; model design /development; mutagen testing; nucleic acid sequence; polymerase chain reaction; skin; tumor suppressor genes

Loss of heterozygosity (LOH) is well documented in cancers. When LOH occurs at tumor suppressor loci, a recessive (mutant) allele becomes functionally homozygous or hemizygous, leading to a relative loss of cellular growth regulation and, ultimately, tumorigenesis. Environment genotoxicants may induce LOH leading to a large fraction of cancers and inherited disease. Thus, it is important to understand mechanisms of LOH and to evaluate environments and agents in terms of their ability to induce LOH in whole animals. Unfortunately, existing whole animal (e.g., transgenic mice) assays do not detect several of the classes of events that produce LOH. Thus, we require practical and sensitive assays that actually detect and quantitate LOH PER SE, independent of mechanism. Using targeted homologous recombination of ES cells, we produced mice that are heterozygous at the adenine phosphoribosyltransferase (Aprt ) locus. In vivo LOH at this locus produces cellular loss of APRT activity, which yields a selectable phenotype when tissues are dissociated and cells cultured. Thus, we have an assay that measures in vivo LOH at an endogenous locus in normal animals. Further, the analysis of polymorphic loci flanking Aprt, Dna sequencing of mutant Aprt genes, and analysis of gene organization and chromosome structure with fluorescent in situ hybridization enables the determination of molecular mechanisms producing LOH. We will examine the spontaneous rate and mechanism of LOH in various mouse tissues, in mice with different genotypes which may predispose cells to LOH and tumorigenesis, and after exposure to genotoxic agents.

杂合性缺失（洛）在癌症中被充分记录。 当洛 发生在肿瘤抑制基因座，隐性（突变）等位基因成为 功能纯合或半合子，导致相对丧失 细胞生长调节和最终的肿瘤发生。 环境 遗传毒物可诱导洛缺失，导致大部分癌症， 遗传性疾病 因此，了解洛缺失的机制是非常重要的 并评估环境和代理人的能力， 整只动物的洛缺失。 不幸的是，现有的整个动物（例如， 转基因小鼠）测定不能检测到 产生洛缺失。 因此，我们需要实用且灵敏的检测方法，实际上 检测和定量洛缺失的PER SE，与机制无关。 使用ES细胞的靶向同源重组，我们产生了 在腺嘌呤磷酸核糖基转移酶（Aprt）基因座是杂合的。 在体内，该位点的洛导致APRT活性的细胞丧失， 当组织解离时产生可选择的表型， 有教养 因此，我们有一种测定体内洛缺失的方法， 正常动物的内源性位点。 此外，多态性分析 Aprt基因侧翼位点，突变Aprt基因的DNA测序，以及Aprt基因的分析。 基因组织和染色体结构与荧光原位 杂交使得能够确定产生 洛。 我们将在不同的细胞中检测洛缺失的自发率和机制。 小鼠组织，在具有不同基因型的小鼠中，可能使细胞 洛缺失和肿瘤发生，以及暴露于遗传毒性剂后。