Targeted Gene Knockouts of p14ARF and p16INK4a

p14ARF 和 p16INK4a 的靶向基因敲除

• 批准号: 6585054
• 负责人: UTZ HERBIG
• 金额: $ 4.81万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6585054
• 关键词: cell growth regulation; cell line; cellular oncology; cytogenetics; gene mutation; gene targeting; immunoprecipitation; molecular pathology; neoplasm /cancer genetics; neoplastic transformation; p53 gene /protein; phosphorylation; polymerase chain reaction; postdoctoral investigator; protein structure function; retinoblastoma protein; southern blotting; transfection; tumor suppressor proteins; western blottings

DESCRIPTION (provided by applicant): One of the most frequently mutated sites found in human tumors is the INK4a/ARF locus located on chromosome 9. This locus encodes the two tumor suppressor proteins p16 INK4a and p14 ARF. Although the function of both proteins in regulating the activities of Rb and p53 is well understood, their contribution in preventing cancer in humans is not. The goal of this proposal is to better understand how loss of p14ARFand/or p16 INK4a function contributes to the formation of tumors in humans. To disrupt p14 ARF and p16 INK4a activity, homozygous knockout cell lines will be generated by targeted homologous recombination in non-transformed, normal human diploid fibroblasts. Targeting vectors will be generated that will specifically disrupt the function of p14ARFand p16 INK4a while leaving the other member of the INK4a/ARF locus intact and functional. The homozygous knockout cell lines will be analyzed on the macroscopic level for proliferation defects and on the molecular level in order to understand and interpret the phenotypic changes observed. The experiments proposed will also determine whether disruption of the p53- and/or the Rb-pathway is required for neoplastic transformation of normal human diploid fibroblasts. p21-/-/p16 INK4a-/-double knockout cell lines will be generated to analyze how the Rb- and p53-pathways are connected on a molecular level and how they cooperate in the prevention of neoplastic transformation of human cells. The data gathered will significantly advance our understanding of how mutations in the INK4/ARF locus contribute to formation of tumors in humans.

描述（由申请人提供）：人类肿瘤中发现的最常见突变位点之一是位于 9 号染色体上的 INK4a/ARF 位点。该位点编码两种肿瘤抑制蛋白 p16 INK4a 和 p14 ARF。尽管这两种蛋白质在调节 Rb 和 p53 活性方面​​的功能已广为人知，但它们在预防人类癌症方面的贡献却尚不清楚。该提案的目的是更好地了解 p14ARF 和/或 p16 INK4a 功能的丧失如何导致人类肿瘤的形成。为了破坏 p14 ARF 和 p16 INK4a 活性，将通过在未转化的正常人二倍体成纤维细胞中进行定向同源重组来产生纯合敲除细胞系。将生成靶向载体，该载体将特异性破坏 p14ARF 和 p16 INK4a 的功能，同时保持 INK4a/ARF 基因座的其他成员完整且功能正常。将在宏观水平上分析纯合敲除细胞系的增殖缺陷，并在分子水平上进行分析，以便理解和解释观察到的表型变化。所提出的实验还将确定正常人二倍体成纤维细胞的肿瘤转化是否需要破坏 p53 和/或 Rb 途径。将产生p21-/-/p16 INK4a-/-双敲除细胞系，以分析Rb-和p53-通路如何在分子水平上连接以及它们如何合作预防人类细胞的肿瘤转化。收集的数据将显着促进我们对 INK4/ARF 位点突变如何促进人类肿瘤形成的理解。