Nada Alduays
Pronouns: She/her
Research Mentor(s): Shelby Hemker
Research Mentor School/College/Department: Human Genetics / Medicine
Program: UROP
Session: Session 3 (11:00am – 11:50am)
Authors: Nada Alduays, Shelby Hemker
Abstract
Loss of function (LOF) in MUTYH results in a high risk of hereditary colorectal cancer. This is mainly because MUTYH is a base excision repair gene that repairs 8-oxo-G:A mispairs in the DNA. The majority of MUTYH variants are VUS (variants of unknown significance), which makes it difficult for clinicians to understand the contribution of each variant to cancer risk. To address this, MAVEs (multiplexed analyses of variant effect) can be used to resolve VUS by the use of high throughput methods. In order to test the function of MUTYH VUSs, we are using a novel GFP-reporter assay. However, there is low transfection efficiency of this GFP-off reporter in cells, making it difficult to measure millions of cells containing different MUTYH VUS at once. To increase the percentage of cells with the reporter, we will clone a new GFP-off reporter assay that includes antibiotic resistance. We are utilizing the newly developed EMMA cloning strategy, which uses Golden Gate cloning, to assemble this reporter, but it requires first using site-directed mutagenesis to make a compatible promoter. To do this, we introduce mutations in primers and PCR amplifies the promoter sequence, changing the ends from EMMA’s position 18 sequence to position 3. We have successfully created this position 3-compatible promotor and are working on assembling the reporter using the EMMA cloning strategy. Future work will include constructing the reporter and testing for improved GFP-off reporter transfection efficiency. These results will aid in making GFP-off reporter cells efficient enough to test thousands of MUTYH VUS without having high rates of cell death.
