NON TUMORIGENIC TERATOCARCINOMA CELL LINE

非致瘤性畸胎癌细胞系

• 批准号: 6206886
• 负责人: PAULETTE J MCCORMICK
• 金额: $ 3.37万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206886
• 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).

胚胎癌细胞(EC)已被证明在 肿瘤发生和哺乳动物发育的分析以及在 对这两个过程相互关联性的研究。特别值得注意的是 这些细胞在转基因动物的发育过程中以及在 维甲酸介导的分化治疗的研究。以前我们 用含有A基因的缺陷型逆转录病毒感染EC细胞株(NR1-0) 新霉素耐药盒(NeoR)，并建立了突变细胞系 编号为NR1-6。该细胞系在形态、粘附性、 致癌和分化特性。突变的遗传分析， 杂交和突变细胞系表明，只有一个 逆转录病毒插入部位调节所有这些隐性的，以及， 显然，遗传上有关联，表型。为了阐明分子 我们已经测序了17.1kb的多效性突变的调控 基因组DNA位于插入部位的两侧。除了重复之外 元素，这个区域与以前的任何 因此是一个新的基因座。使用各种不同的 我们搜索了在该区域和 在基因组的其他地方，其表达可能是直接或间接的 受插入位点位基因调控。 我们已经确定了至少三个新的外显子编码在17kb内 侧翼序列，其中两个与3.3kb的转录本杂交。 对小鼠细胞和组织进行Northern分析。使用差异显示 和信使核糖核酸指纹图，我们还确定了一些潜在的 NR1-6和NR1-0之间表达差异的下游靶基因 细胞系。我们现在建议更详细地评估这些发现，见 为了了解调节原始NR1-6的分子机制 并找出调节基因突变的下游作用基因 不同的表型(尤其是对 维甲酸)与NR1-6突变有关。为此，我们将进行 进一步的分子和功能分析：S编码的转录本 插入基因座(特异性靶点1)，以及由其他基因座编码的转录本 已证明它们在亲本和亲本之间有差异表达 突变细胞(特异性AIM2)。