Targeted Gene Knockouts of p14ARF and p16INK4a

描述（由申请人提供）：在人类肿瘤中发现的最常见的突变位点之一是位于9号染色体上的INK4a/ARF位点。该基因座编码两种肿瘤抑制蛋白p16 INK4a和p14 ARF。虽然这两种蛋白在调节Rb和p53活性方面的功能已经被很好地理解，但它们在预防人类癌症方面的作用还不清楚。该提案的目标是更好地了解p14ARFand/或p16 INK4a功能的丧失如何促进人类肿瘤的形成。为了破坏p14 ARF和p16 INK4a活性，将在未转化的正常人类二倍体成纤维细胞中通过靶向同源重组产生纯合敲除细胞系。将产生靶向载体，特异性地破坏p14ARFand p16 INK4a的功能，同时使INK4a/ARF位点的其他成员保持完整和功能。纯合子敲除细胞系将在宏观水平和分子水平上分析增殖缺陷，以理解和解释所观察到的表型变化。提出的实验还将确定p53-和/或rb通路的破坏是否需要正常人类二倍体成纤维细胞的肿瘤转化。将生成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，以及它们如何协同预防人类细胞的肿瘤转化。收集到的数据将极大地促进我们对INK4/ARF基因座突变如何促进人类肿瘤形成的理解。

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.