Analysis of Gene Function by Parallel Phenotyping using Barcoded Mutants

使用条形码突变体通过平行表型分析基因功能

• 批准号: 7858219
• 负责人: ADAM KUSPA
• 金额: $ 32.31万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858219
• 关键词: Activities of Daily Living; Antineoplastic Agents; Ants; Biological; Cell Adhesion; Cells; Cisplatin; Cloning; Code; DNA; DNA Sequence; Deposition; Development; Dictyostelium; Drops; Escherichia coli; Future; G-Protein-Coupled Receptors; Genes; Genomics; Germination; Growth; Growth and Development function; Haploidy; Individual; Insertion Mutation; Knock-out; Laboratories; Measures; Mediating; Methods; Molecular; Mutagenesis; Mutate; Mutation; Natural Immunity; Nucleotides; Oligonucleotide Microarrays; Oligonucleotides; Organism; Phenotype; Plasmids; Population; Population Control; Protein Kinase; Proteins; Publishing; Relative (related person); Research; Research Personnel; Resistance; Sampling; Site; Staging; Testing; Time; base; cohort; digital imaging; fitness; functional genomics; gene function; homologous recombination; interest; knockout gene; migration; mutant; plasmid DNA; programs; receptor; research study; restriction enzyme; slug; transcription factor; web site

The proposed research plan is part of a long-term program aimed at understanding the molecular mechanisms that control development in Dictyostelium. We aim to mutate genes and study the resulting phenotypes as an avenue to discovering the function these genes. By using a combination of random mutagenesis and directed knockout strategies we will generate mutations in about half of the 10,500 protein coding genes where each mutation is tagged with a unique 60-nucleotide DNA sequence (a molecular barcode). Insertion mutations will be induced in a haploid strain (AX4) by restriction enzyme mediated ntegration (REMI) of plasmid DNA, selected at random, and cloned by plasmid rescue. The DNA sequence flanking each clone, and therefore the insertion site of each mutation, will be determined and the genomic ocations of the insertions will be published to the project website (dictygenome.org) for distribution of the mutants ant the knockout plasmids. We will also use a PCR-based method that we have developed to knockout selected cohorts of genes, such as those encoding protein kinases, transcription factors and putative cell adhesion and recognition receptors. As one measure of gene function, we will determine the ability of each mutant to carryout various developmental and growth-stage functions in mixtures of 768 mutants. In these competitive phenotyping experiments, each mutant will be detected by hybridization of its barcode DNA tag to a barcode oligonucleotide microarray, after PCR amplification of all barcodes in the mixture. For example, a complete set of mutants will be taken through several cycles of growth, development, sporulation and germination, and DNA samples will be made from the mutants surviving each successive step. Mutants that drop out of this population, but persist in a control population of cells that were propagated without intervening cycles of development will be recorded as developmentally defective. Additional experiments that sub-divide development into definable steps (aggregation, slug migration, etc.) will further narrow the mutant phenotypes. We will carryout additional functional tests using these parallel analysis methods and, when appropriate, by phenotyping individual mutants. Integrating these results with the results of the transcriptional phenotyping (Project II) will allow us to propose regulatory networks (Project III) that can be tested by future experiments.

拟议的研究计划是旨在了解分子生物学的长期计划的一部分 控制盘基网柄菌发育的机制。我们的目标是突变基因并研究由此产生的结果 表型作为发现这些基因功能的途径。通过使用随机组合 诱变和定向敲除策略我们将在 10,500 个蛋白质中的大约一半产生突变 编码基因，其中每个突变都标有独特的 60 核苷酸 DNA 序列（分子 条形码）。插入突变将通过限制酶介导在单倍体菌株（AX4）中诱导 随机选择质粒 DNA 的整合 (REMI)，并通过质粒拯救进行克隆。 DNA序列 每个克隆的侧翼，以及每个突变的插入位点，都将被确定，并且基因组 插入的位置将发布到项目网站（dictygenome.org）以供分发 突变体和敲除质粒。我们还将使用我们开发的基于 PCR 的方法 敲除选定的基因组，例如编码蛋白激酶、转录因子和 假定的细胞粘附和识别受体。作为基因功能的一种衡量标准，我们将确定 第768章 突变体。在这些竞争性表型实验中，每个突变体将通过其杂交来检测 对条码寡核苷酸微阵列中的所有条码进行 PCR 扩增后，将条码 DNA 标签添加到条码寡核苷酸微阵列上 混合物。例如，一套完整的突变体将经历几个生长周期， 发育、孢子形成和萌发，并且将从每个存活的突变体中提取 DNA 样本。 连续的步骤。突变体从该群体中消失，但仍保留在对照细胞群体中， 在没有干预发育周期的情况下进行繁殖将被记录为发育缺陷。 将发育细分为可定义步骤的其他实验（聚合、slug 迁移等） 将进一步缩小突变表型。我们将使用这些并行进行额外的功能测试 分析方法，并在适当时对个体突变体进行表型分析。将这些结果与 转录表型分析（项目 II）的结果将使我们能够提出调控网络（项目 III) 可以通过未来的实验来检验。