Project Team
Students
Adrien Chen
Chemistry
Penn State Harrisburg
Faculty Mentors
Nik Tsotakos
Penn State Harrisburg
Biological sciences
Esther Gomez
Penn State University Park
Chemical engineering
Project
Project Video
Project Abstract
MC REU 2022 ABSTRACT
Adrien Chen
Penn State Harrisburg
Major: Chemistry
Anticipated graduation date: May 2025
Primary faculty mentor: Dr. Nik Tsotakos (Penn State Harrisburg)
Secondary faculty mentor: Dr. Esther Gomez (Penn State University Park)
Engineering of a podocyte cell line expressing MEG3 using CRISPRa.
Human glomerular epithelial cells (HGEC), also known as podocytes, are important cells forming the filtration apparatus in our kidneys. Approximately one third of people having diabetes develop a complication known as diabetic kidney disease during which podocytes lose their structure and function. Previous experiments have shown that high glucose can lead to podocyte damage. It was found that a long non-coding RNA (lncRNA) called maternally expressed gene 3 (MEG3), is involved in glucose-induced podocyte injury. However, the molecular etiology by which MEG3 functions in podocytes is unknown. Creating a new cell line expressing MEG3 in a normal glucose environment can result in a novel tool to investigate the effect of MEG3 on the podocytes. In order to create the cell line, we used CRISPR-activation (CRISPRa) to induce endogenous MEG3 expression in the previously established HGEC cell line. We infected the cells with lentivirus carrying the endonuclease dead Cas9 -VP64 transactivation domain fusion gene (dCas9-VP64) and selected by puromycin treatment. The selection didn’t yield many viable cells meaning that the transduction was not highly efficient. The reason for this may be that the virus concentration obtained was too low. In order to troubleshoot this step, quantitative polymerase chain reaction (qPCR) can be performed to verify the virus concentration. Once created, this novel CRISPRa MEG3 expressed HGEC cell line can serve as a tool for scientists to discover the mechanism by which MEG3 impairs the podocyte. Better understanding of lncRNA MEG3 in podocytes may lead to better treatment for people diagnosed with diabetic kidney disease.
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