DERIVATION OF REVERTANTS OF RAS-TRANSFORMED HUMAN CELLS

RAS 转化人类细胞回复体的衍生

• 批准号: 3459311
• 负责人: LEONARD E BENADE
• 金额: $ 9.71万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3459311
• 关键词: athymic mouse; cell bank /registry; cell transformation; gene interaction; genetic mapping; helper virus; human tissue; hybrid cells; molecular cloning; mouse sarcoma virus; murine leukemia virus; mutant; natural gene amplification; neoplasm /cancer genetics; neoplastic cell; nitrosoguanidines; nucleic acid sequence; oncogenes; oncogenic virus; ouabain; regulatory gene; transfection; viral rescue

Revertants of ras-transformed cells will be derived from the KHOS cell line. These cells, transformed by a single copy of the Kirsten murine sarcoma virus (KiMuSV) genome, were derived from HOS cells, which were originally cloned from a human osteosarcoma. Although it is a continuous, immortalized human line, HOS exhibits none of the recognized properties of transformed cells. It is readily transformed, however, by various oncogenic viruses and by chemical means. A second Ki-ras gene has been introduced into the KHOS cells to reduce the frequency of revertants due to alterations of the transforming gene itself. Following nitrosoguanidine mutation, flat revertants will be selected using ouabain treatment. KHOS is much more highly susceptible to ouabain than non- transformed, parental HOS cells. Revertant cells thus obtained will be assayed-for anchorage-independent growth in soft agar and tumorigenicity in nude mice. Those lines failing to exhibit these transformed characteristics will then be examined for active transforming genes by rescuing transforming virus from KHOS revertants using amphotropic murine leukemia virus (MuLV) as a helper and assaying focus-forming activity on NIH 3T3 cells. The presence of the ras gene product p21 will be confirmed by Western (protein) blotting. Those revertant lines which can be demonstrated to have retained active transforming genes will be tested for re-transformation by ras-containing Harvey and Kirsten viruses. We will also perform cell fusions with other transformed lines to look for dominance of transformation suppression in the resulting hybrids. In making hybrids with mouse cell lines we will attempt to map the loci responsible for suppression to specific human chromosomes. Finally, we will try to isolate the sequence(s) involved in suppression by transfecting KiMuSV-transformed NIH 3T3 cells, once again using ouabain to select for revertants.

Ras转化细胞的逆转体将从khos中获得。 细胞系。这些细胞，由单个拷贝的 柯尔斯滕鼠肉瘤病毒(KiMuSV)基因组，来源于HOS 这些细胞最初是从人类骨肉瘤中克隆出来的。 虽然这是一条连续的、永垂不朽的人类路线，但居屋展示了 转化的细胞没有任何公认的特性。它是 然而，容易被各种致癌病毒和由 化学手段。第二个Ki-ras基因已经被导入到 KHOS细胞减少因改变而产生的返回体的频率 转化基因本身。在亚硝胺之后 突变后，将使用哇巴因处理选择扁平回复突变体。 Khos比非khos更容易感染哇巴因 转化的亲代HOS细胞。这样获得的反转细胞 将在软琼脂和软琼脂中进行非锚定生长的检测 裸鼠体内的致瘤性。那些没有表现出这些的线条 然后将检查转换后的特征是否处于活动状态 从KHO中抢救转化病毒进行基因转化 利用两性小鼠白血病病毒(MuLV)作为 辅助和检测NIH3T3细胞的成灶活性。这个 Ras基因产物p21的存在将被Western证实 (蛋白质)印迹。那些回复的线条可以是 被证明保留了活跃的转化基因将是 通过包含ras的哈维和柯尔斯滕进行重新转换测试 病毒。我们还将与其他转化的细胞进行细胞融合 中寻找变换抑制的主导地位的行 由此产生的混血儿。在与小鼠细胞系杂交的过程中，我们将 尝试将负责抑制的基因座映射到特定的 人类的染色体。最后，我们将尝试分离序列(S) KiMuSV转化的NIH3T3细胞参与抑制 细胞，再一次使用哇巴因来选择返回体。