Toward a Complete Set of Germline Null and Modifiable Mutations in Drosophila

果蝇中一整套种系无效突变和可修饰突变

基本信息

批准号：
8779682
负责人：
Ming Fa
金额：
$ 21.97万
依托单位：
GENETIVISION CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8779682
关键词：
Address Alleles Animal Model Applied Research Basic Science Biological Models Biological Process Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities Complement Custom DNA DNA Transposable Elements Development Docking Drosophila genus Drosophila melanogaster Engineering Evaluation Excision Fusion Protein Expression Gene Expression Profiling Gene Targeting Generations Genes Genetic Genetic Complementation Test Genetic Recombination Genome Genomics Goals Human Human Biology Human Development Injection of therapeutic agent Integrase Knowledge Letters Mammals Mediating Messenger RNA Methods Modification Molecular Molecular Genetics Mutagenesis Mutation Pattern Phase Phosphorus Point Mutation Proteins Protocols documentation Reagent Research Research Personnel Resources Site Source Staging System Technology Testing Time Transcript Transgenic Organisms arm base design flexibility fly gene complementation gene function genome sequencing homologous recombination insight interest loss of function loss of function mutation mutant new technology novel novel strategies public health relevance research study screening success tool

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this proposal is to generate, maintain, and distribute a new collection of engineered Drosophila melanogaster stocks that will allow versatile manipulation of genes for which there is currently no mutant allele available. Using cutting edge CRISPR technology, this collection or 'kit' will represent a powerful new set of tools that will benefit virtually all Drosophila geneticists, and it is likely that there will b a high demand for this resource for many years. Many of the most important advances in our understanding of human development have come from studies using Drosophila as an animal model system. Since many parallels exist between Drosophila and mammals in terms of the underlying molecular mechanisms controlling biological processes, knowledge gained from research in Drosophila can be either directly applied or readily adapted to understanding human biology. One key factor that sets Drosophila apart from other model systems is the huge wealth of genetic and molecular tools that have accumulated in the past 100 years of research. To study any given gene, two essential reagents are loss-of-function mutations and a tagged version of the gene. Currently available methods to obtain mutant or tagged alleles of genes cover only a portion of the genome or are inefficient, laborious, and time consuming. Indeed, there are more than 2000 genes in the fly genome that currently lack a mutant allele with no means of efficiently generating either a loss-of-function mutant or a tagged allele. To address this problem, we propose to generate a new targeted collection that will complement existing collections and offer the ultimate flexibility of downstream manipulation, such as generating both point and loss-of-function mutations, protein tagging, and gene expression profiling. We plan to split this project into two phases: Phase I will serve as a proof of principle and optimize the conditions for CRISPR- mediated targeting while Phase II will generate a collection targeting 2000 genes currently without a mutation. To achieve this goal, we will: Aim 1. Develop and optimize CRISPR-Cas9 mediated cassette targeting. Aim 2. Evaluate CRISPR-mediated cassette targeting across the genome. Success in Phase I will develop robust protocols for CRISPR-mediated cassette targeting and set the stage for expanding our effort to generate a complete collection during Phase II. Harnessing the benefits of new technologies, this collection will be another key addition to the Drosophila research tool kit. As a sign of the strong enthusiasm this proposal has received from many prominent Drosophila researchers, a set of 11 letters of support are included with this application.

描述（由申请人提供）：该提案的总体目标是生成，维护和分发新的工程果蝇Melanogaster股票集合，该股票将允许对当前没有突变等位基因的基因进行多功能操纵。使用Cust Edge CRISPR技术，该系列或“套件”将代表一套有力的新工具，这些工具几乎将使所有果蝇遗传学家受益，并且很可能有很多年的资源需求很高。我们对人类发展的理解中许多最重要的进步都来自使用果蝇作为动物模型系统的研究。由于果蝇和哺乳动物之间存在许多相似之处，从控制生物学过程的基本分子机制方面，因此可以直接应用果蝇研究获得的知识，也可以很容易地适应理解人类生物学。将果蝇与其他模型系统区分开的关键因素是在过去100年的研究中积累的大量遗传和分子工具。为了研究任何给定的基因，两种基本试剂是功能丧失突变和标记的基因。当前获得基因突变或标记等位基因的方法仅涵盖基因组的一部分或效率低下且耗时。实际上，蝇基因组中有超过2000个基因缺乏突变等位基因，没有有效产生功能丧失的突变体或标记的等位基因。为了解决这个问题，我们建议生成一个新的目标集合，该集合将补充现有收集，并提供下游操纵的最终灵活性，例如产生功能丧失突变，蛋白质标记和基因表达分析。我们计划将该项目分为两个阶段：第一阶段将作为原理的证明，并优化了CRISPR介导的靶向条件，而第二阶段将产生针对目前没有突变的2000个基因的集合。为了实现这一目标，我们将：目标1。开发和优化CRISPR-CAS9介导的盒式定位。 AIM 2。评估CRISPR介导的盒子靶向整个基因组。第一阶段的成功将为CRISPR介导的盒式目标制定强大的协议，并为扩大我们在II期期间产生完整收集的努力奠定了基础。利用新技术的好处，该系列将是果蝇研究工具套件的另一个关键补充。为了表明该提议从许多著名的果蝇研究人员那里得到的强烈热情，本申请中包括一组11个支持信。