ANALYSIS OF A NON-TUMORIGENIC TERATOCARCINOMA CELL LINE

非致瘤性畸胎癌细胞系的分析

• 批准号: 3193573
• 负责人: PAULETTE J MCCORMICK
• 金额: $ 12.41万
• 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193573
• 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是特别有价值的研究 肿瘤发生和哺乳动物发育以及评估 这两种现象之间的关系。我们已经感染了胚胎 用逆转录病毒感染癌细胞， 突变导致细胞的分化缺陷， 或致癌潜力。一个这样的细胞系，最初通过其 独特的形态表型，在两个参数方面都是异常的。 这些细胞不沿沿着典型的胚胎癌干分化 细胞谱系可能已经失去了他们的能力， 衍生物.最重要的是，它们也失去了形成 同基因小鼠的肿瘤。Southern印迹分析表明， 变异细胞系具有单个病毒插入物，而原始细胞是 可能是半合子的此外，我们最近 分离出一种回复突变细胞系， 逆转录病毒的自发切除。因此，如果变体确实是 插入突变的结果，一个基因可以调节两个 细胞的致瘤性和分化能力。是 本建议的目的是探讨这一可能性， 克隆这个基因为此目的，将侧翼于所述多核苷酸的基因组序列插入到所述多核苷酸的基因组序列中。 将分离前病毒。在这个序列中， 鉴定出显示亲本细胞和变体细胞之间的差异。 分离相应的cDNA并用于DNA转染 检测插入和表型之间因果关系的试验 变化或者，两种细胞之间差异表达的基因 将通过差异cDNA克隆的方法分离细胞系， 类似的分析。父母的分化潜力和 将在体外利用两种方法检查和比较变异细胞系， 分子、生物化学和免疫学标记技术和体内 通过囊胚注射嵌合体的生产。最后， 与变异表型相关的生化改变将被 表征了对所获结果的分析肯定应加强 我们对细胞和分子元素的了解 分化和肿瘤发生以及它们之间的可能联系 这两种现象。