Novel transgenesis and expression technology for nematodes

线虫新型转基因和表达技术

• 批准号: 10186102
• 负责人: MICHAEL L NONET
• 金额: $ 26.99万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/10186102
• 关键词: 3' Untranslated Regions; Adopted; Adoption; Animal Model; 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; FLP recombinase; Frequencies; Gene Expression Regulation; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Engineering; Genetic Recombination; Genome; Genomics; Germ Cells; Grant; Health; Human Biology; Mediating; Methods; Modeling; Modification; Molecular; Monitor; Nematoda; Nobel Prize; Nuclear; Oligonucleotides; Phenotype; Play; Point Mutation; Procedures; Publications; RNA Interference; RNA Viruses; Regulation; Reporter; Research; Research Personnel; Resources; Role; Scientist; Signaling Molecule; Site; System; Systems Integration; Techniques; Technology; Testing; Tetanus Helper Peptide; Toxin; Transgenes; Transgenic Animals; Transgenic Organisms; Work; cell type; comparative efficacy; design; experimental study; flexibility; improved; insight; new technology; novel; novel strategies; optogenetics; plasmid DNA; programs; promoter; protein degradation; recombinase; recombinase-mediated cassette exchange; repaired; sensor; technique development; time use; tool; vector; virtual

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 技术在几乎所有使用 DNA 的研究项目中都发挥着不可或缺的作用。 线虫模型剖析介导人类许多方面的保守生物机制 健康和疾病生物学。虽然 CRISPR 技术的发展彻底改变了 科学家有能力对基因组进行小幅修改，创造出含有以下成分的转基因动物： 大型的数千碱基插入仍然很费力。这些类型的转基因动物是 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，标记和可视化亚细胞成分，监测信号分子的浓度 使用基因编码传感器，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型重组酶介导的 线虫盒交换方法可将转基因频率提高约五倍 超过当前的技术。此外，我使用这项新技术开发了四个双向报告器 在线虫中使用的系统，以促进转基因工具的稳健表达。虽然小说 方法是对当前方法的重大改进，它仍然大于一个数量级 效率比 CRISPR 技术低很多。开发过程中的见解 技术指出了本次赠款旨在克服的关键限制，以进一步提高效率 的方法。此外，由于使用 Flp，新方法具有很大的局限性 和 Cre 重组酶。这笔赠款还建议进一步技术开发该方法 克服这些限制。拟议工作的成功实施将产生极大的影响 通过极大地促进转基因开发对秀丽隐杆线虫研究界产生影响 这是许多研究项目的共同瓶颈。