CAREER: Tagged Transposon-Mediated Mutagenesis in C.elegans

职业生涯：线虫中标记的转座子介导的突变

• 批准号: 9733685
• 负责人: Erik Jorgensen
• 金额: $ 49.41万
• 依托单位: University of Utah
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9733685&HistoricalAwards=false
• 关键词: CAREER; Tagged; Transposon; Mediated; Mutagenesis

Jorgensen 9733685 Abstract Exocytosis in neurons has become specialized in order to allow for the rapid secretion of neurotransmitter. A number of proteins involved in synaptic vesicle trafficking have been identified by biochemical purification from synaptosomes. Nevertheless, current models for neurosecretion are greatly limited because there is only an incomplete list of the proteins that are required for these processes. Genetic studies in the nematode, Caenorhabditis elegans, could potentially identify the complete complement of genes required for synaptic function. C. elegans is particularly advantageous for such studies because it is possible to select for mutants defective in secretion. Using such screens, over 38 genes have been identified which are required for normal neurotransmission. The efficiency of these screens has generated more mutants than can be easily characterized using standard genetic techniques. Techniques to rapidly map and clone these genes lags far behind the ability to obtain mutants. Mutagenesis strategies using transposons modified to contain foreign plasmid sequences could allow the immediate cloning of mutant genes without the need to map the mutations. The aims of this proposal are to: 1) Develop methods to mobilize recombinant Tc3 transposons in the germ line, 2) Develop techniques to mobilize a heterologous mariner element in C. elegans, 3) Train undergraduates to screen for neurotransmission mutants in C. elegans using a recombinant mariner element and to directly sequence the mutant genes in a single semester course. The tools developed by this work will benefit the larger scientific community and accelerate experiments designed to analyze neurotransmission. The techniques developed will greatly increase the efficiency of forward genetic methods. Additionally, the development of these tools and reagents will provide students with a more complete laboratory experience.

乔根森9733685抽象胞吐作用在神经元中已经变得专门化，以允许神经递质的快速分泌。通过对突触小体进行生化纯化，已鉴定出许多与突触小泡运输有关的蛋白质。然而，目前的神经分泌模型非常有限，因为只有这些过程所需的蛋白质的不完整清单。对线虫--秀丽线虫的遗传学研究，可能会确定突触功能所需的全部基因。线虫对这类研究特别有利，因为有可能选择分泌缺陷的突变体。使用这样的筛查，已经识别出了38个正常神经传递所需的基因。这些筛选的效率已经产生了比使用标准基因技术容易描述的更多的突变。快速定位和克隆这些基因的技术远远落后于获得突变体的能力。使用含有外源质粒序列的转座子的诱变策略可以立即克隆突变基因，而不需要绘制突变图谱。这项建议的目的是：1)开发在胚系中动员重组TC3转座子的方法；2)开发技术来动员线虫中的异源水手元件；3)培训本科生使用重组水手元件来筛选线虫的神经传递突变，并在一个学期的课程中直接对突变基因进行测序。这项工作开发的工具将造福于更大的科学界，并加快旨在分析神经传递的实验。开发的技术将极大地提高正向遗传方法的效率。此外，这些工具和试剂的开发将为学生提供更完整的实验室体验。