Zoe Gargiulo
Research Mentor(s): Roberto Marquez Pizano
Research Mentor School/College/Department: EEB
Presentation Date: 08/03/2022
Presentation Type: Poster
Poster Number: 1
Session: Session I: 12:30 – 1:20pm
Room: League Ballroom
Authors: Zoe Gargiulo, Christopher Talbot, Roberto Marquez
Abstract
CRISPR/Cas9 genome editing technologies have opened up the field of genetic research to a broader range of study systems. Developing new techniques for genetic engineering in non-model amphibians requires changing many aspects of the standard protocol found in model systems. A crucial step in performing Cas9-CRISPR genetic modification on amphibians is dejellying the embryos – removing the layers of protective protein enveloping the embryos to allow for microinjection without clogging or breaking the injection needle. Two standard practices exist for dejellying an amphibian embryo – manually removing the jelly using tweezers, or soaking the embryos in a chemical solution that breaks down the surrounding layers of jelly. Manual dejellying introduces the risk of human error, which may result in inviable embryos. Even successful manual dejellying may expose the embryos to excessive physical disturbance, impacting long-term survivorship. Chemical dejellying, on the other hand, must be performed very precisely. Unsuccessful chemical dejellying may destroy entire clutches of embryos if the dejellying agent reaches the embryo membrane and begins to break it down. This study aims to evaluate the overall survivorship rates of these two different treatments to identify the best way to prepare embryos for injection with minimal harm. Once we have identified the ideal dejellying technique for survivorship, we can begin varying other parameters of the Cas9-CRISPR process, such as injection techniques and Cas9-CRISPR concentration, to optimize survivorship.
