EAGER: Overcoming transformation and germline expression barriers

University Of Kansas Center For Research Inc, Lawrence KS

Investigators

Abstract

A cell’s genome, comprised of DNA, enables cells to function and respond to changing environments. Seminal experiments by Frederick Griffith in the 1920s revealed that some bacterial cells capture DNA from the environment (released from dying cells). The cells incorporated the captured DNA into their genomes where it functioned normally. Historically, this easy ability to acquire DNA has not been observed in plant and animal cells. From this, a common view is that plant and animal cells actively protect their genomes from invasion of foreign, or virus-derived, DNA encountered from the environment, and many genome-protection strategies are known. These protective mechanisms prevent the efficient introduction of DNA into cells in the laboratory setting where scientists might aim to correct a genetic defect or perform other genetic manipulations in cells. The goal of this project is to investigate delivery and intracellular trafficking routes for DNA introduced to cells from environmental sources using the genetically tractable, multicellular organism Caenorhabditis elegans. In addition to acquiring knowledge, new strategies for genetic manipulation and DNA delivery are predicted outcomes from this project. Undergraduate students will derive broader benefits from the project as experiments will be reserved for them. Small amounts of various DNA fragments can be detected in the circulatory system of animals and plants. Detection and analysis of circulating DNA (circDNA) for the purpose of diagnosing disease, and the impact of circDNA on cell function, is an emerging and expanding field of research. circDNA activates inflammation responses in higher organisms and has been observed to incorporate into distant cells. How frequently circDNA manages to gain entry into distant cells and incorporate into that cell’s genome is a relatively unexplored question. Using Caenorhabditis elegans, we will investigate protein-mediated delivery of circDNA to distal cells. C. elegans lacks many inflammatory pathways found in higher eukaryotes, such as cGAS-STING and NF-kappaB nuclear effector responses. Thus, we can more easily and directly investigate the consequences of exposure to circulating, extracellular DNA and the consequences of its acquisition to cells. If successful, this EAGER project will provide new strategies for genetic manipulation through purposeful introduction of DNA. We may also learn of additional cell or developmental consequences to circDNA, consequences that may be avoided due to circDNA-related inflammatory responses in higher organisms. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.