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的长期计划的一部分 控制dictyostelium的发展的机制。我们旨在突变基因并研究结果 表型是发现这些基因功能的途径。通过随机组合 诱变和定向敲除策略我们将在10,500个蛋白质中的一半中产生突变 编码基因，其中每个突变都用独特的60-核苷酸DNA序列标记（分子 条形码）。插入突变将通过限制酶介导的单倍体菌株（AX4）诱导 质粒DNA的NTEGRATION（REMI），随机选择，并由质粒救援克隆。 DNA序列 将确定每个克隆的侧面，因此将确定每个突变的插入位点，并确定基因组 插入的插入将发布到项目网站（dictygenome.org）进行分发 突变蚂蚁敲除质粒。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 推定的细胞粘附和识别受体。作为基因功能的一种度量，我们将确定 每个突变体在768的混合物中携带各种发育和生长阶段功能的能力 突变体。在这些竞争表型实验中，将通过其杂交检测每个突变体 在PCR扩增所有条形码之后，条形码DNA标签是寡核苷酸微阵列 混合物。例如，将通过几个生长循环进行完整的突变体， 开发，孢子形成和发芽以及DNA样品将由每个生存的突变体制成 连续的步骤。从该人群中辍学的突变体，但仍在对照的细胞群中存在 在没有干预开发周期的情况下进行传播将被记录为发育中有缺陷。 将开发分为可确定的步骤（聚集，sl迁移等）的其他实验 将进一步缩小突变表型。我们将使用这些并行进行其他功能测试 分析方法，并在适当的情况下通过表型分型来进行单个突变体。将这些结果与 转录表型的结果（项目II）将使我们能够提出监管网络（项目 iii）可以通过将来的实验来测试。