CAREER: Tagged Transposon-Mediated Mutagenesis in C.elegans

摘要神经元的胞吐作用已成为一种特化的机制，以促进神经递质的快速分泌。许多参与突触囊泡运输的蛋白质已经通过生化纯化从突触体中鉴定出来。然而，目前的神经分泌模型非常有限，因为这些过程所需的蛋白质只有一个不完整的列表。对秀丽隐杆线虫（Caenorhabditis elegans）的遗传学研究，有可能鉴定出突触功能所需的完整基因。秀丽隐杆线虫对这类研究特别有利，因为它有可能选择分泌缺陷的突变体。通过这种筛选，已经确定了超过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表征的更多的突变体。快速绘制和克隆这些基因的技术远远落后于获得突变体的能力。利用含有外源质粒序列的转座子进行修饰的诱变策略可以立即克隆突变基因，而无需绘制突变图谱。本课题的目标是：1)开发在种系中调动重组Tc3转座子的方法；2)开发在秀丽隐杆线虫中调动异源mariner元件的技术；3)培养本科生使用重组mariner元件筛选秀丽隐杆线虫的神经传递突变体，并在单学期课程中直接对突变基因进行测序。这项工作开发的工具将使更大的科学界受益，并加速旨在分析神经传递的实验。所开发的技术将大大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。

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.