Novel transgenesis and expression technology for nematodes

重组DNA技术在几乎所有使用DNA的研究项目中都起着不可或缺的作用

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.