ANALYSIS OF IN VITRO DIFFERENTIATION OF A HUMAN HEPATOMA

人肝癌细胞系Hep G2在体外分化，从一个细胞系向另一个细胞系发展。 胎儿肝脏的表型特征更接近于 成年人 这种差异是由一个戏剧性的透镜表示， 培养物的倍增时间， 甲胎蛋白、醛缩酶A、C和丙酮酸激酶K， 白蛋白、醛缩酶B和丙酮酸激酶L的合成。 这项建议 描述了在此期间白蛋白基因的详细分析， 分化 信使RNA水平和转录的变化将是 使用北方印迹法、核径流实验和定量 溶液杂交测定。 这项研究的第二阶段涉及 构建杂合基因并转染到Hep G2中以确定 负责分化依赖性改变的序列。 瞬态 表达测定将使用假定的肝特异性 连接到CAT的调控序列，CAT是一种细菌基因， 标记。 这应该可以合理快速地描绘序列 白蛋白表达所必需的。 补充实验将进行监测 在具有不同拷贝的稳定转染子中的杂合基因处的转录 number. 这项工作的第三阶段将使用无细胞转录 从Hep G2（一种亚克隆）制备的提取物，用于鉴定蛋白质 肝特异性白蛋白基因转录因子 表情

The human hepatoma line Hep G2 differentiates in vitro, progressing from a phenotype characteristic of fetal liver to one which more closely resembles the adult. This differentiation is denoted by a dramatic lenthening of the doubling time of the cultures, a decline in the synthesis of alphafetoprotein, aldolase A, C and pyruvate kinase K and an increase in the synthesis of albumin, aldolase B and pyruvate kinase L. This proposal describes a detailed analysis of the albumin gene during this differentiation. Messenger RNA levels and changes in transcription will be measured using Northern blots, nuclear run off experiments and quantitative solution hybridization assays. A second phase of this study involves the construction of hybrid genes and transfection into Hep G2 to define the sequences responsible for differentiation dependent alterations. Transient expression assays will be carried out using putative liver specific regulatory sequences attached to CAT, a bacterial gene to be used as a marker. This should allow a reasonably rapid delineation of the sequences necessary for albumin expression. Complementary experiments will monitor transcription at the hybrid gene in stable transfectants with varying copy number. A third phase of this work will use cell free transcription extracts prepared from Hep G2, a subclone, to identify protein transcriptional factors responsible for liver specific albumin gene expression.