NON TUMORIGENIC TERATOCARCINOMA CELL LINE

非致瘤性畸胎癌细胞系

• 批准号: 2856292
• 负责人: PAULETTE J MCCORMICK
• 金额: $ 23.29万
• 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 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2856292
• 关键词: RNase protection assay; Retroviridae; athymic mouse; cell differentiation; complementary DNA; embryonic stem cell; gene expression; gene mutation; gene targeting; genetic library; genetic regulation; genetic regulatory element; genetically modified animals; messenger RNA; mutant; northern blottings; nucleic acid hybridization; nucleic acid sequence; phenotype; polymerase chain reaction; teratoma; tissue /cell culture; transfection; transposon /insertion element; viral carcinogenesis

Embryonal carcinoma (EC) cells have proven to be of tremendous value in analyses of both oncogenesis and mammalian development as well as in studies of the interrelatedness of these two processes. Of particular note are the use of these cells in the development of transgenic animals and in studies of retinoid mediated differentiation therapy. Previously we infected an EC cell line (NR1-0) with a defective retrovirus containing a neomycin resistance cassette (NeoR) and created a mutant cell line designated NR1-6. This cell line is unique in its morphological, adhesive, tumorigenic and differentiative properties. Genetic analyses of mutant, hybrid and revertant cell lines indicate that there is only a single retroviral insertion site regulating all of these recessive, and, apparently, genetically linked, phenotypess. To elucidate the molecular regulation of this pleiotropic mutation we have sequenced 17.1 kb of genomic DNA flanking the insertion site. With the exception of repeat elements, this region shares no significant homology with any previously reported sequence and is therefore a novel locus. Using a variety of techniques we searched for transcripts encoded both within this region and elsewhere in the genome whose expression might be directly or indirectly regulated by the insertion site locus. We have identified at least three novel exons encoded within the 17 kb flanking sequence, two of which hybridize to a transcript of 3.3 kb in northern analysis of mouse cells and tissues. Using differential display and mRNA fingerprinting we have also identified a number of potential downstream target genes whose expression differ between the NR1-6 and NR1-0 cell lines. We now propose to evaluate these findings in more detail in order to understand the molecular mechanisms regulating the original NR1-6 mutation and to identify the downstream acting loci which regulate the variant phenotypes (most particularly differentiative response to retinoids) associated with the NR1-6 mutation. To this end we will conduct further molecular and functional analyses of: the transcript(s) encoded by the insertion locus (Specific Aim 1), and transcripts encoded by other loci which have been shown to be differentially expressed between parent and mutant cells (Specific Aim2).

胚胎癌（EC）细胞已被证明在治疗癌症方面具有巨大价值。 分析肿瘤发生和哺乳动物发育以及 研究这两个过程的相互关系。 特别值得注意的 这些细胞在转基因动物的发展中的应用， 类维生素A介导的分化治疗研究。 以前我们 感染EC细胞系（NR 1 -0），用含有 新霉素抗性盒（NeoR），并创建突变细胞系 命名为NR 1 -6。 该细胞系在形态学、粘附性、 致瘤性和分化特性。 突变体的遗传分析， 杂交和回复突变细胞系表明， 逆转录病毒插入位点调节所有这些隐性的，和， 很明显，遗传连锁，表现型。 为了阐明 我们已经测序了17.1 kb的 插入位点侧翼的基因组DNA。 除了重复 元件，该区域与任何先前的序列没有显著的同源性。 报道的序列，因此是一个新的基因座。 使用各种 技术，我们搜索了在这个区域内编码的转录本， 在基因组的其他地方，其表达可能直接或间接 由插入位点基因座调节。 我们已经确定了至少三个新的外显子编码的17 kb内， 侧翼序列，其中两个与3.3kb的转录本杂交， 小鼠细胞和组织的北方分析。 使用差分显示 和mRNA指纹图谱，我们也发现了一些潜在的 在NR 1 -6和NR 1 -0之间表达不同的下游靶基因 细胞系 我们现在建议更详细地评估这些发现， 为了了解调节原始NR 1 -6的分子机制， 突变，并确定下游作用基因座，调节 变异表型（最特别的是对 与NR 1 -6突变相关的类维生素A）。 为此，我们将 进一步的分子和功能分析： 插入基因座（特异性目的1）和由其他基因座编码的转录物 已经显示其在亲本和亲本之间差异表达， 突变细胞（特异性Aim 2）。