Targeted Gene Knockouts of p14ARF and p16INK4a

描述（申请人提供）：人类肿瘤中最常见的突变位点之一是位于9号染色体上的INK 4a/ARF基因座。该基因座编码两种肿瘤抑制蛋白p16 INK 4a和p14 ARF。虽然这两种蛋白质在调节Rb和p53活性方面的功能已经很好地理解，但它们在预防人类癌症方面的贡献还不清楚。该提案的目的是更好地理解p14 ARF和/或p16 INK 4a功能的丧失如何有助于人类肿瘤的形成。为了破坏p14 ARF和p16 INK 4a活性，将在非转化的正常人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，其将特异性地破坏p14 ARF和p16 INK 4a的功能，同时使INK 4a/ARF基因座的其他成员保持完整和功能性。将在宏观水平上分析纯合敲除细胞系的增殖缺陷，并在分子水平上分析纯合敲除细胞系，以了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p53-和/或Rb-通路的破坏。将产生p21-/-/p16 INK 4a-/-双敲除细胞系，以分析Rb-和p53-通路如何在分子水平上连接，以及它们如何在预防人类细胞的肿瘤转化中合作。收集的数据将大大促进我们对INK 4/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.