Analyses of a Non tumorigenic Teratocarcinoma Cell Line

描述：我们已经使用逆转录病毒插入创造了一个突变的胚胎 肿瘤(EC)细胞系NRI-6，在形态、黏附、 致癌和分化特性。突变体、杂交种的遗传分析 而突变的细胞系表明只有一种逆转录病毒 我们已经映射到小鼠X的近端的插入部位 染色体。小鼠乳腺表达序列Taq(EST)具有大于 与插入位点侧翼18kb的区域有97%的同源性 我们已经测序的基因组DNA。利用这种EST进行分子研究， 我们已经确定了两个在亲本中表达的转录本，但在突变体中没有表达， 细胞。主要转录本-2.3kb包含两个外显子，第二个外显子 其被插入而被破坏。表情分析表明，这是 转录产物在时间和空间上都有广泛的表达。我们假设 这份成绩单的丢失是NRI-6的根本基础 I突变和这个转录本在两个胚胎中都起着关键作用 发育和成人的动态平衡。 我们还寻找了其他可能作用于基因下游的基因 调节与基因相关的特定表型的插入位点 突变(即下游效应基因)。我们已经发现，核子 受体RARI3和y在较高的基础水平表达 突变细胞与亲本细胞比较及组蛋白抑制 脱乙酰基使亲本水平增加到突变体的特征水平 细胞。 我们还发现，组蛋白脱乙酰酶的抑制对 突变体与亲本细胞生物学的其他关键参数。我们假设 突变细胞具有较低的组蛋白脱乙酰酶活性，与某些特定的 启动子(特别是RARB和y)比亲本细胞更多，而且 因此，这些核受体的表达增加，至少 部分原因是观察到的突变型视黄醇过敏症。 最后，我们分离并分析了另一个假定的下游效应器 一种名为MyoR的基因，在突变细胞中相对于亲本细胞高表达 或者是复原者。该基因编码一种新的碱性螺旋-环-螺旋(BLHLH) 转录因子曾被认为是一种抑制因子 胚胎骨骼肌肌发生。然而，我们发现，表达 在非常早期的胚胎(3 5dpc I囊胚)中，ECIES细胞 表达MyoR的基因既不分化为骨骼肌，也不表达 专有肌源性转录因子(即MyoD、myf5)。这些结果产生了 这让我们假设MyoR在早期发挥着更广泛、更基础的作用 胚胎发生比之前怀疑的要多。 在本提案中，我们将对上述三个假设进行评估。我们 将分离、克隆、测序和翻译全长插入基因座 转录本，确定其基因组结构和调控，并检测 蛋白质产品的功能(S)(具体目标一)。我们将继续我们的 分析突变细胞的维甲酸超敏反应，我们的努力集中在 组蛋白乙酰化的作用，特别是关于RARB和Y 推动者。(具体目标二)。最后，我们将研究MyoR的调节 并检测其在早期胚胎发育中的表达和作用。 NRI-6突变[特异靶III]。这些研究将做出重大贡献 有助于我们了解干细胞生物学、胚胎发育和癌症。

DESCRIPTION: We have used retroviral insertion to create a mutant embryonal carcinoma (EC) cell line, NRI-6, that is unique in its morphological, adhesive, tumorigenic and differentiative properties. Genetic analyses of mutant, hybrid and revertant cell lines indicates that there is only a single retroviral insertion site which we have mapped to the proximal portion of the mouse X chromosome. A mouse mammary gland expressed sequence taq (EST) has greater than 97 percent homology to a region within the >18kb of insertion site flanking genomic DNA that we have sequenced. Utilizing this EST for molecular studies, we have identified two transcripts expressed in parental, but not in mutant, cells. The predominant transcript, -2.3kb, contains two exons, the second of which is disrupted by the insertion. Expression analyses indicates that this transcript is widely expressed both temporally and spatially. We hypothesize that loss of this transcript is the underlying basis for the NRI-6 I mutation and that this transcript plays a critical role in both embryonic development and adult homeostasis. We have also searched for other genes that might act downstream of the insertion site locus to regulate specific phenotypes associated with the mutation (i.e., downstream effector genes). We have found that the nuclear receptors RARI3 and y are expressed at higher basal levels in mutant cells as compared to parental and that inhibition of histone deacetylation increases parental levels to those characteristic of mutant cells. We have also found that histone deacetylase inhibition differentially affects other key parameters of mutant vs parental cell biology. We hypothesize that mutant cells have less histone deacetylase activity associated with certain promoters (specifically RARB and y) than do parental cells and that the consequent increased expression of these nuclear receptors accounts, at least in part, for the observed mutant retinoid hypersensitivity. Finally, we have isolated and analyzed another putative downstream effector gene called MyoR that is highly expressed in mutant cells relative to parental or revertant. This gene encodes a novel basic helix-loop-helix (bLHLH) transcription factor that had been proposed to function as a repressor of embryonic skeletal muscle myogenesis. However, we have found that is expressed in very early stage embryos (3 5dpc I blastocyst) and that the ECIES cells which express MyoR neither differentiate into skeletal muscle nor express the obligate myogenic transcription factors (i.e., MyoD, myf5). These results have led us to hypothesize that MyoR plays a broader, more fundamental role in early embryogenesis than was previously suspected. In this proposal, we will evaluate the three hypothesis proffered above. We will isolate, clone, sequence and translate the full length insertion locus transcript, determine its genomic structure and regulation and examine the function of the protein product(s) (Specific Aim I). We will continue our analyses of the mutant cell retinoid hypersensitivity, focusing our efforts on the role of histone acetylation, specifically as regards the RARB and y promoters. (Specific Aim II). Finally, we will study the regulation of the MyoR gene and examine its expression and role in early embryogenesis and in the NRI-6 mutation [Specific Aim III]. These studies will contribute significantly to our understanding of stem cell biology, embryonic development and cancer.