期刊
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS
卷 1866, 期 9, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.bbalip.2021.158991
关键词
Triacylglycerol; Gene editing; Acyltransferase; CRISPR/Cas9; Endoplasmic reticulum; Homology-directed repair
资金
- College of Medicine (University of Saskatchewan)
- Canada Foundation for Innovation
The study utilized CRISPR/Cas9 gene editing to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells, allowing successful detection of the protein at its endogenous locus within the cell.
In eukaryotic organisms, two unrelated acyl-CoA:diacylglyceml acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the final step of the triacylglycerol biosynthetic pathway. Both enzymes are highly expressed in lipogenic tissues, such as adipose tissue, small intestine and the liver. DGAT2 has a prominent role in hepatocyte lipid metabolism synthesizing triacylglycerols that are utilized for very low-density lipoprotein assembly. However, due to the lack of useful antibodies to detect endogenous DGAT2 protein, it has been difficult to determine how this enzyme functions at the cellular level. We have unsuccessfully tested many commercial antibodies as well as our own in-house antibodies. There is currently no evidence that DGAT2 undergoes processing such that antigenic epitopes to these antibodies are removed. As an alternative, many studies have utilized epitope tagged versions of DGAT2 overexpressed in cells. These approaches can provide valuable information about a protein, but can be subject to artifacts, such as mislocalization, misregulation, protein aggregation and abnormal protein-protein interactions. In this study, we used gene editing with CRISPR/Cas9 to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells. HepG2 cells, derived from a human hepatocellular carcinoma, have been routinely used as a cell model to study human hepatocyte lipid and lipoprotein metabolism. Using this system allowed us to successfully detect DGAT2 expressed from its endogenous locus in HepG2 cells by immunoblotting with anti-FLAG antibodies.
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