Thesis Defense Announcement
To: The George Mason University Community
Candidate: Cindy T. Duong
Program: M.S. in Biology
Date: Monday November 30, 2015
Time: 3:00 PM
Place: George Mason University
Science & Tech (Prince William) Campus
Institute for Advanced Biomedical Research (IABR), Room 1004
Title: "Development and Optimization of Novel Tools for Exploring RNA-Based Cell-Cell Communication "
Thesis Director: Dr. Claudius Mueller
Committee Chair: Dr. Lance Liotta
Committee Members: Dr. Alessandra Luchini, Dr. Ancha Baranova
A copy of the thesis will be available in the Gateway Library. All are invited to attend the defense.
Cells communicate by sending and receiving messages that are necessary to carry out essential functions. In cancer, this intricate signaling network of interactions may help tumor cells develop resistance to treatment that could potentially lead to patient death. There are currently no available tools to observe how tumor cells communicate in vivo. Here, we developed two tools to visualize RNA-based cell-cell communication, using hybridization chain reactions (HCR) to kill cells that communicate and a Cre-lox gene construct to cause communicating cells to fluoresce.
HCR that exhibited strong polymerization was based on hairpin 1 and 2 (H1/2) loop, stem, toe and overall length, as well as trigger oligonucleotide concentration. However, transfection with HCR components including and excluding a trigger
oligonucleotide induced cell death. Plasmids containing target HCR component sequences flanked by a 5’-hammerhead ribozyme (HHR) and a 3’-Hepatitis Delta Virus (HDV) ribozyme were Created to enable endogenous production of HCR components in cells. In vitro
transcription of plasmids indicated ribozyme cleavage and release of target RNA. The H1, H2, and I RNA produced after transcribing the individual plasmids yielded HCR.
Plasmids containing Cre recombinase, ribozyme, LoxP-STOP-LoxP, and green fluorescent protein (GFP) were Created to permanently record cell-cell communication. The original gene construct with the start codon (ATG) in front of GFP caused
strong false positive fluorescence in cells, while repositioning the ATG in front of LoxP-STOP-LoxP showed a dramatic decrease in fluorescence. To further reduce background fluorescence, a new plasmid was built with HHR at both the 5’- and 3’-end of Cre to
ensure Cre is cleaved and not made into a protein. Inhibitors complementary to the stemloops of each HHR were predicted in silico and tested, resulting in one candidate displaying ribozyme cleavage inhibition.
Our studies demonstrate the ability of the HCR mechanism to mediate cell death in cultured human cancer cells. We further developed a Cre recombinase gene construct flanked by two ribozymes that can be specifically inhibited using a short trigger oligonucleotide. Both technologies show promise to become critical tools for visualizing RNA-based cell-cell communication and will be developed further in the future.