ANALYSIS OF A NON-TUMORIGENIC TERATOCARCINOMA CELL LINE

非致瘤性畸胎癌细胞系的分析

• 批准号: 3193577
• 负责人: PAULETTE J MCCORMICK
• 金额: $ 16.56万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193577
• 关键词: DNA; RNA; Retroviridae disease; antibody; carcinogenesis; cell differentiation; clone cells; early embryonic stage; gene expression; gene mutation; genetic library; genetic manipulation; genetic transcription; genome; laboratory mouse; molecular cloning; teratoma; transfection; transposon /insertion element

Embryonal carcinoma cells have proven to be of particular value in studies of both oncogenesis and mammalian development as well as in evaluating the relationship between these two phenomena. We have infected embryonal carcinoma cells with a retrovirus in an effort to create insertion mutations which result in cells defective in either their differentiative or oncogenic potentials. One such cell line, originally identified by its unique morphological phenotype, is abnormal in respect to both parameters. These cells do not differentiate along typical embryonal carcinoma stem cell lineages possibly having lost their ability to elaborate endodermal derivatives. Most significantly, they have also lost their ability to form tumors in syngeneic mice. Southern blot analysis indicates that this variant cell line has a single viral insert and the original cell was probably hemizygous for the insertion site. In addition we have recently isolated a revertant cell line which appears to have arisen through spontaneous excision of the retrovirus. Therefore, if the variant is indeed a consequence of an insertional mutation, a single gene may regulate both the tumorigenic and differentiative capacities of the cell. It is the purpose of this proposal to explore this possibility by identifying and cloning this particular gene. To this end the genomic sequence flanking the provirus will be isolated. Within the sequence, a transcript will be identified that shows a difference between the parental and variant cells. The corresponding cDNA will be isolated and used in a DNA transfection assay to examine the causal relationship between insertion and phenotypic change. Alternatively, genes differentially expressed between the two cell lines will be isolated by the method of differential-cDNA cloning and similarly analyzed. The differentiative potentials of the parental and variant cell lines will be examined and compared both in vitro utilizing molecular, biochemical and immunological marking techniques and in vivo through the production of blastocyst injection chimeras. Eventually, biochemical alterations associated with the variant phenotypes will be characterized. Analyses of the results obtained should certainly enhance our knowledge of the cellular and molecular elements controlling differentiation and oncogenesis as well as the possible connection between these two phenomena.

胚胎癌细胞已被证明在研究中具有特殊的价值 肿瘤发生和哺乳动物发育以及在评估 这两种现象之间的关系。我们已经感染了胚胎 携带逆转录病毒的癌细胞努力创造插入 导致细胞在分化过程中出现缺陷的突变 或致癌潜能。一个这样的细胞系，最初由它的 独特的形态表型，在这两个参数上都是异常的。 这些细胞不会沿着典型的胚胎癌干细胞分化 细胞谱系可能已经失去了精致内胚层的能力 衍生品。最重要的是，它们也失去了形成的能力 同基因小鼠的肿瘤。Southern杂交分析表明，这是 变异细胞系只有一个病毒插入，而原始细胞是 可能是插入部位的半合子。此外，我们最近还 分离了一株似乎是通过 逆转录病毒的自发切除。因此，如果变种确实是 作为插入突变的结果，单个基因可能调节两者 细胞的致瘤和分化能力。它是 这项建议的目的是通过确定和确定 克隆这种特殊的基因。为此，位于基因两侧的基因组序列 前病毒将被隔离。在序列中，一份成绩单将 经鉴定，表明亲本细胞和变异细胞之间存在差异。 相应的cdna将被分离并用于DNA转染。 检测插入和表型之间因果关系的方法 变化。或者，在两个细胞之间差异表达的基因 将采用差异克隆的方法分离品系，并 类似的分析。亲本和亲本的分化潜力 变异的细胞系将在体外进行检测和比较 分子、生化和免疫学标记技术及体内研究 通过生产胚泡注射嵌合体。最终， 与变异表型相关的生化改变将是 特色化的。对所获得的结果的分析肯定应该加强 我们对控制细胞和分子元素的知识 分化与肿瘤发生及其可能的联系 这两种现象。