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Novel transgenesis and expression technology for nematodes

线虫新型转基因和表达技术

基本信息

批准号：
10620801
负责人：
MICHAEL L NONET
金额：
$ 27.56万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10620801
关键词：
3&apos Untranslated Regions Adopted Adoption Animals Apoptosis Binding Sites Biological Biological Models CRISPR/Cas technology Caenorhabditis elegans Cell Nucleus Cell physiology Communities Cre-LoxP DNA DNA Sequence DNA cassette DNA delivery Data Development Disease Drosophila genus Elements Enterobacteria phage P1 Cre recombinase Event Exclusion FLP recombinase Frequencies Gene Expression Regulation Gene Transfer Techniques Generations Genes Genetic Genetic Engineering Genome Genomics Germ Cells Grant Health Human Biology Mediating Methods Modeling Modification Molecular Monitor Natural regeneration Nematoda Nobel Prize Nuclear Oligonucleotides Phenotype Play Point Mutation Procedures Publications RNA Interference RNA Viruses Regulation Reporter Reporter Genes Research Research Personnel Resources Role Scientist Signaling Molecule Site System Systems Integration Techniques Technology Testing Tetanus Helper Peptide Toxin Transgenes Transgenic Animals Transgenic Organisms Visualization Work cell type comparative efficacy design experimental study flexibility improved insight model organism new technology novel novel strategies optogenetics plasmid DNA programs promoter protein degradation recombinase recombinase-mediated cassette exchange repaired sensor technique development technology development time use tool transgene expression vector virtual

项目摘要

Recombinant DNA technology plays an integral role in virtually every research program using the C. elegans model to dissect conserved biological mechanisms that mediate many aspects of human biology in health and disease. While the development of CRISPR technology has revolutionized the ability of scientists to make small modification of the genome, creating transgenic animals that contain large multi-kilobase inserts remains laborious. These types of transgenic animals are required for many critical aspects of dissecting cellular mechanisms including to express genes in specific cell types, to tag and visualize sub-cellular components, to monitor concentrations of signaling molecules using genetically encoded sensors, and to perturb cellular functions using RNA interference and selective protein degradation technologies. I have recently developed a novel recombinase-mediated cassette exchange approach for C. elegans that increases the frequency of transgenesis about five-fold over current techniques. Furthermore, I used this novel technology to develop four bipartite reporter systems for use in nematodes to facilitate robust expression of transgenic tools. While the novel approach is a significant improvement over current approaches, it remains greater than an order of magnitude less efficient than CRISPR technology. Insights made during the development of the technique point to critical limitations that this grant aims to overcome to further increase the efficiency of the approach. Furthermore, the new approach comes with significant limitations due to the use of Flp and Cre recombinases. This grant also proposes further technological development of the approach to overcome these limitations. Successful implementation of the proposed work would have extreme impact on the C. elegans research community by greatly facilitating transgene development removing this common bottleneck for many research programs.

重组DNA技术在几乎每个研究项目中都发挥着不可或缺的作用秀丽线虫模型剖析调节人类许多方面的保守生物机制健康与疾病中的生物学。虽然CRISPR技术的发展彻底改变了科学家对基因组进行微小修改的能力，创造出含有大的几千碱基插入仍然很费力。这些类型的转基因动物是必需的剖析细胞机制的许多关键方面，包括在特定细胞中表达基因类型，以标记和可视化亚细胞组件，以监测信号分子的浓度使用基因编码的传感器，并使用RNA干扰和干扰细胞功能选择性蛋白质降解技术。我最近开发了一种新的重组酶介导的用于线虫的盒式交换方法将转基因频率提高约五倍过多的现有技术。此外，我还利用这一新技术开发了四个双方记者在线虫中使用的系统，以促进转基因工具的稳健表达。虽然这部小说方法是对当前方法的重大改进，它仍然大于比CRISPR技术低得多的效率。在发展过程中提出的见解技术指出了这项拨款旨在克服的关键限制，以进一步提高这种方法。此外，由于FLP的使用，新方法也有很大的局限性和Cre重组酶。这笔赠款还建议进一步开发方法的技术，以克服这些限制。拟议工作的成功实施将具有极端的极大地促进转基因开发移除对线虫研究界的影响这是许多研究项目的共同瓶颈。