GENERATION OF DEVELOPMENT MUTANTS WITH CLONED DNA VECTOR

使用克隆 DNA 载体产生发育突变体

• 批准号: 3479321
• 负责人: HAROLD M WEINTRAUB
• 金额: $ 31.81万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3479321
• 关键词: Anura; cell differentiation; cell growth regulation; cell type; chickens; complementary DNA; developmental genetics; gene expression; genetic manipulation; granulocyte; hematopoietic stem cells; macrophage; molecular cloning; mutant; myogenesis; nucleic acid hybridization; nucleic acid sequence; recombinant DNA

The overall goal of this proposal is to try to identify genes responsible for determining cell type specificity and eventually to try to understand the genetic complexity and logic of how this specificity is generated. We will be focusing on four types of developmental systems: early decision making in specific frog lineages; genes that commit cells to myogenesis; genes that determine whether hematopoietic precursor cells differentiate into macrophages or granulocytes; and genes that seem to link developmental decisions to decisions of growth control. For many of these systems, we begin by using cDNA clones that are cell type or cell lineage specific and we try to elicit a "phenotype" by introducing these sequences back into cells -- in a positive sense, to induce commitment with normal expression of the vector; in a negative sense, to inhibit differentiation with a vector that produces "abnormal" transcripts. We are hoping to create recombinant DNA mutations using 4 approaches. All of these methods must yield dominant mutations to be useful. The first is to use anti-sense RNA; the second is to use standard in vitro mutagenesis of cloned DNA to create a dominant mutation; the third is to overproduce the gene product; the fourth is to express a tissue-specific gene in the wrong cell type. For all of these schemes we will restrict our attention to tissue or lineage specific sequences obtained by "substractive" cloning of the unique RNAs (or cDNAs) peculiar to a given cell type. In most cases, this material is readily available. Estimates from solution hybridization suggest that if the two cell types that are to be compared are closely related, one might expect between 200-1000 sequences in such a difference library. These could be screened either in pools or individually. In the case of red blood cells, the screen is to look for embryos that lack red cells.

该提案的总体目标是尝试确定负责的基因 用于确定细胞类型特异性，并最终尝试理解 这种特异性的产生方式的遗传复杂性和逻辑。 我们 将重点关注四种类型的发展系统：早期决策 制作特定的青蛙谱系；将细胞引起肌发生的基因； 确定造血前体细胞是否有区别的基因 进入巨噬细胞或粒细胞；和似乎连接发展的基因 决策增长控制的决定。 对于许多这样的系统，我们 首先使用特定于细胞类型或细胞谱系的cDNA克隆，然后 我们试图通过将这些序列引入回到中来引起“表型” 细胞 - 从积极的意义上诱导正常表达的承诺 矢量；从负面意义上讲，抑制与 产生“异常”转录本的向量。 我们希望使用4种方法创建重组DNA突变。 全部 这些方法必须产生主要突变才能有用。 第一个是 使用抗敏感RNA；第二个是使用标准的体外诱变 克隆的DNA产生显性突变；第三个是生产过量 基因产物；第四是在该组织中表达组织特异性基因 错误的单元格类型。 对于所有这些方案，我们将限制我们的注意力 通过“扣除”克隆获得的组织或谱系特定序列 给定细胞类型的独特RNA（或cDNA）的独特之处。 大多数 情况，此材料很容易获得。 从解决方案估计 杂交表明，如果要比较两种细胞类型 是密切相关的，人们可能会期望在这种A中的200-1000个序列之间 差异库。 这些可以在池中筛选，也可以 单独。 对于红细胞，屏幕要寻找 缺乏红细胞的胚胎。