ROLE OF PROTO-ONCOGENES IN MAMMALIAN EMBRYOGENESIS

原癌基因在哺乳动物胚胎发生中的作用

• 批准号: 3330983
• 负责人: THOMAS W SADLER
• 金额: $ 13.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330983
• 关键词: antisense nucleic acid; biotechnology; cell biology; cytogenetics; developmental genetics; embryo /fetus tissue /cell culture; embryogenesis; gene expression; genetic regulatory element; genetic techniques; immunocytochemistry; laboratory rat; microinjections; protein structure function; protooncogene; regulatory gene; scanning electron microscopy

The proto-oncogenes are thought to play key roles in mammalian embryogenesis. However, data supporting this hypothesis are circumstantial and are based on investigations showing correlations between expression of genes and morphological events, and not on gene function. While homologous recombination in ES cells combined with transgenic mice technology has created gene "knockouts" to determine proto-oncogene function; this technique is laborious and incapable of assessing gene function at specific points in time and space during development. Therefore we have developed a method to specifically inhibit the expression of individual genes in space and time using whole mammalian embryo culture. Normal development of mouse embryos is supported in vitro and antisense oligonucleotides are microinjected into the amniotic cavity and specific tissues. With this system, morphological phenotypes, including altered brain and cardiac development, associated with inhibition of Wnt-1 proto-oncogene expression have been determined. The defects are induced only by antisense probes and not by sense oligo's. Furthermore, their induction is restricted to injections made at the time of gene expression in the hindbrain region of neurulating embryos (5 somite stage: day 9; plug day = day 1). We propose to expand these results and to pursue the function of two other proto-oncogenes N-myc and ax1. These 3 proto-oncogenes were selected because their general pattern of expression has been determined in mouse embryos and suggests important roles for different aspects of organogenesis. Furthermore, the different spatial and temporal characteristics of their expression provide an opportunity to expand the technical capacity of antisense inhibition in studying murine organogenesis. The specific aims will be: 1) to better define our understanding of when these 3 genes are expressed using in situ hybridization and immunocytochemistry; 2) to determine the effects of inhibiting expression of these 3 proto-oncogenes at specific stages of development using antisense probes. 3) to determine the biological effects of antisense inhibition of the proto-oncogene expression at the cellular level.

原始基因被认为在哺乳动物中起关键作用 胚胎发生。 但是，支持此假设的数据是 间接和基于显示相关性的调查 在基因的表达和形态事件之间，而不是基因上 功能。 而ES细胞中的同源重组 转基因小鼠技术创建了基因“敲除”以确定 原始癌基因函数；这项技术是费力的，无能为力 在特定时间和空间中评估基因功能 发展。 因此，我们已经开发了一种方法来具体 使用整体抑制各个基因在空间和时间上的表达 哺乳动物胚胎培养。 小鼠胚胎的正常发育是 体外和反义寡核苷酸的支持 羊水和特定组织。 使用此系统， 形态学表型，包括大脑和心脏的改变 开发，与抑制Wnt-1原型癌基因有关 表达已确定。 缺陷仅由 反义探测，而不是从意义上讲。 此外，它们的归纳 仅限于在基因表达时进行的注射 神经胚胎的后脑区域（5个Somite阶段：第9天；塞日 =第1天）。 我们建议扩大这些结果并追求功能 其他两个原始基因N-MYC和AX1。 这3种原始基因是 之所以选择，是因为它们的一般表达方式已确定 在小鼠胚胎中，提出了重要的作用 器官发生。 此外，不同的空间和时间 他们表达的特征提供了扩展的机会 反义抑制的技术能力在研究鼠 器官发生。 具体目标是：1）更好地定义我们的 了解何时使用原位表达这三个基因 杂交和免疫细胞化学； 2）确定 在特定阶段的特定阶段抑制这3种原始癌基因的表达 使用反义探针开发。 3）确定生物学 反义抑制原型癌基因在 细胞水平。