Yashwanth Bajji
Pronouns: he/him
Research Mentor(s): Xin Tong
Research Mentor School/College/Department: Molecular & Integrative Physiology / Medicine
Program:
Authors: Xin Tong
Session: Session 1: 9:00 am – 9:50 am
Poster: 93
Abstract
E4BP4 is a gene that acts as a transcription factor in the liver originally found to regulate circadian rhythms, but now is more heavily studied for its role in immune cell development and function. E4BP4 has also been found to promote lipid droplet formation in the liver in nonalcoholic fatty liver disease (1). Crispr/cas9 is a gene editing system that allows us to use the cas9 protein as “genetic scissors” to edit genes for our needs. Here, we will use a GFP-reporter system to validate the editing of the E4bp4 gene by Crispr/cas9. We insert a gene for the GFP protein, EGFP, into the plasmid, then insert an E4BP4 specific gene sequence in the middle of the EGFP gene. The gRNA used for the Crispr/cas9 system will be specific to the E4BP4 gene sequence. Thus, if the Crispr/cas9 works as intended, the E4BP4 sequence will be cut out by cas9 and the EGFP gene will be transcripted, resulting in the GFP protein being expressed. Expression of the GFP protein leads to fluorescence under green light fluorescence microscopy. We found that when a mammalian cell was transfected with the EGxxFP and cas9/gRNA blueprint plasmids, the resulting Crispr edited cells fluoresced under UV light, indicating GFP expression. This validation system can be used to check the efficiency of the Crispr/cas9 system on the E4BP4 gene sequence. (1) Wang S, Yang M, Li P, Sit J, Wong A, Rodrigues K, Lank D, Zhang D, Zhang K, Yin L, Tong X. High-Fat Diet-Induced DeSUMOylation of E4BP4 Promotes Lipid Droplet Biogenesis and Liver Steatosis in Mice. Diabetes. 2023 Mar 1;72(3):348-361. doi: 10.2337/db22-0332. PMID: 36508222; PMCID: PMC9935497.
