Targeted Gene Knockouts of p14ARF and p16INK4a

描述(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基因座。该基因座编码两种肿瘤抑制蛋白p16INK4a和p14ARF。虽然这两种蛋白质在调节Rb和P53活性方面的功能已被熟知，但它们在预防人类癌症方面的贡献还不是很清楚。这项建议的目的是为了更好地了解p14ARF和/或p16INK4a功能缺失是如何导致人类肿瘤的形成。为了破坏p14ARF和p16INK4a的活性，将在未转化的正常人类二倍体成纤维细胞中通过定向同源重组产生纯合子敲除细胞系。靶向载体将被产生，它将特别干扰p14ARF和p16INK4a的功能，同时保持INK4a/ARF基因座的另一个成员完整和起作用。为了理解和解释观察到的表型变化，将在宏观水平上分析纯合子敲除细胞系的增殖缺陷，并在分子水平上进行分析。建议的实验还将确定是否需要破坏P53和/或RB途径来实现正常人类二倍体成纤维细胞的肿瘤性转化。将产生p21-/-/p16 INK4a-/-双基因敲除细胞系，以分析Rb-和P53-通路是如何在分子水平上连接的，以及它们如何在防止人类细胞的肿瘤转化中协同作用。收集的数据将极大地促进我们对INK4/ARF基因突变如何导致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.