TRANS-ACTIVATING FACTORS AND GLOBIN GENE EXPRESSION--A DIRECT APPROACH

反式激活因子和珠蛋白基因表达——直接方法

• 批准号: 3940485
• 负责人: H DAVE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3940485
• 关键词: DNA; cell bank /registry; developmental genetics; gene expression; genetic manipulation; genetic transcription; hormone regulation /control mechanism; human tissue; messenger RNA; mouse mammary tumor virus; neoplastic cell culture for noncancer research; nucleic acid sequence; oncogenes; plasmids; transforming virus

Humans undergo two developmental switches in their hemoglobin phenotype. The embryonic to fetal switch early in gestation and the fetal to adult switch around the time of birth. The K562 human leukemia cell line expresses all globin genes other than the adult beta-globin. Previous work from this laboratory has shown that the K562 beta-globin gene functions normally in a heterologous expression system. Elucidation of the mechanism of failure of beta-globin gene expression in K562 cells may provide an insight into globin gene expression and switching in normal erythroid cells. The direct isolation of trans-activating gene(s) will be attempted using the strategy that led to the isolation of several oncogenes. Hybrid beta-Neo plasmids, which do not express in K562 cells, will be cotransfected with another selectable marker (RSV-GPT). Stable tranformants will be obtained by selecting for GPT and the presence of beta-Neo confirmed by Southern blotting. High molecular weight genomic DNA from K562 and MEL cells will be transfected into these clones and the activation of beta-Neo sought by G418 selection. The genomic DNA will be fractionated until the gene(s) of interest is/are isolated. A "rescue" strategy will be used when studying MEL cell genomic DNA. C-myc has been studied as putative trans-acting factor for beta and epsilon globin genes. Expression was not detected in heterologous transient assay systems using CAT activity as a marker. Further studies using stable K562 cell transformants containing c-myc are in progress as are studies looking for a potential repressor effect of c-myc. No suitable human cell lines expressing beta globin are available. It has not proven possible to immortalize marrow erythroid progenitors using combinations of c-myc, c-Ha-ras, and E1A oncogenes. This is felt to reflect suboptimal transfection and/or growth conditions resulting from a scarcity of human material. Optimization will be attempted using murine marrow prior to returning to human marrow studies.

人类的血红蛋白经历了两个发育开关