Date of Award


Document Type



Santa Clara University

First Advisor

Leilani M. Miller


Genetic engineering has always held great opportunity for the field of gene therapy. If the cause of a genetic disease can be determined, correction of this gene would allow for an efficient and permanent cure. However, current technologies utilizing engineered retroviruses have serious drawbacks that significantly limit their practical applications for gene therapy. In this project we investigate a novel genetic editing technology called TALENs. By utilizing a modular protein isolated from a plant pathogen that can be quickly and efficiently redesigned to recognize and bind to any desired sequence of DNA combined with a nuclease, we can target a double stranded break at any location in the genome, setting off a cascade of events we can exploit to insert a new sequence of DNA at the site of the double stranded break. While the goal is to develop the TALENs technology for use on humans, extensive research is needed before human trials can be conducted. Our group is testing this in the model organism C. elegans, a soil nematode used for genetic studies. One advantage of doing this is the ability to microinject fluid into the gonad of a young adult worm, which will be absorbed by forming oocytes (eggs) during development. Thus any genetic change will be permanent in the subsequent generations. This fluid will contain two important components: mRNA that will code of the TALENs protein to create the double stranded break at the desired location, and a DNA segment that contains homology, or matching base pairs on both sides of the double stranded break, allowing the cell to repair itself with the provided template. We will be tagging our particular gene of interest, lin-39, with GFP to visualize its expression while simultaneously demonstrating the ability to utilize TALENs technology to insert a full gene into the genome. We have successfully created the mRNA and template components to inject into C. elegans. Continued work on this project will produce a useful GFP fusion protein for the Miller lab and will also demonstrate a viable genetic engineering technology that will eventually facilitate gene therapy.