Confirmation and Effects of VACM‐1/Cul5 Gene Knockout in T47D Breast Cancer Cells and HUVEC Cells Using CRISPR‐Cas9 Approach

Abstract

The VACM‐1/Cul5 protein is a part of the ubiquitin E3 ligase system responsible for ubiquitin‐dependent protein degradation. The VACM‐1/Cul5 dependent E3 ligase decreases cellular proliferation, and lack of regulation in this pathway can lead to cancer. The CRISPR‐Cas9 system is a bacterial immune system that functions by targeting specific sequences of DNA. It can be programmed to target genes of interest and enables specific gene editing in eukaryotic cells. This system was previously used to knockout VACM‐1/Cul5 in a T47D breast cancer cell line and in a human umbilical vein endothelial cell line (HUVEC). The immunostaining results of control and CRISPR‐transfected HUVEC and T47D cells indicate knockout of VACM‐1. Confirmation of the knockout in the T47D cell line was achieved through a T7 Endonuclease 1 mismatch cleavage assay, proving a homozygous mutation of both alleles. AlamarBlue® growth assays in the T47D cell line and Matrigel® growth assays in the HUVEC cell lines show that VACM‐1/Cul5 knockouts in both cell lines increase proliferation. Together, these results suggest that VACM‐1/CUL5 is an important regulator of cellular growth in breast cancer and endothelial cells. Our current work focuses on determining the effects of the VACM‐1/Cul5 knockout on T47D and HUVEC cell signaling pathways.Support or Funding InformationThis work was supported by the Sherman Fairchild Research Grant to S. Bonema and Biology Department.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.