ECI2 Knockout 786-O Polyclonal Cells comprise a heterogeneous polyclonal cell population derived from the 786-O human clear cell renal cell carcinoma (ccRCC) line, engineered via CRISPR/Cas9-mediated disruption of the ECI2 gene. This product provides a loss-of-function model for studying enoyl-CoA delta isomerase 2, an enzyme critical for the isomerization of unsaturated fatty acyl-CoA esters during mitochondrial and peroxisomal beta-oxidation. The polyclonal knockout cells are supplied as a pool of edited cells, eliminating time-consuming single-cell cloning and enabling immediate application in functional assays. This format retains the genetic diversity inherent to polyclonal editing while achieving robust target-gene inactivation across the population.
The parental 786-O cell line is a widely used model for ccRCC, derived from a primary clear cell adenocarcinoma of the kidney. These cells harbor a VHL gene mutation resulting in loss of the von Hippel-Lindau tumor suppressor protein (pVHL), leading to constitutive stabilization of hypoxia-inducible factor 2-alpha (HIF-2??) under normoxia. This genetic background drives the pseudohypoxic transcriptional program characteristic of ccRCC, including altered metabolic gene expression. The 786-O line is extensively employed to investigate HIF-2???Cdependent oncogenic mechanisms and therapeutic vulnerabilities in kidney cancer.
ECI2 encodes mitochondrial and peroxisomal isoforms of enoyl-CoA delta isomerase 2, which catalyze the conversion of 3-cis- and 2-trans-enoyl-CoA esters to the 2-trans-enoyl-CoA intermediates required for subsequent hydration and dehydrogenation in the beta-oxidation spiral. ECI2 activity is transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR-??) and its coactivator PGC-1??, and is responsive to AMP-activated protein kinase (AMPK) signaling. In VHL-deficient ccRCC, HIF-2?? contributes to the regulation of ECI2 expression, linking oxygen sensing to lipid catabolism. ECI2 functions upstream of the production of acetyl-CoA, NADH, FADH2, and ATP, and supports ketone body formation during fasting. It physically interacts with the peroxisomal bifunctional enzyme EHHADH and the mitochondrial trifunctional protein HADHA, and requires the peroxisomal targeting receptor PEX5 for peroxisomal import. The enzyme operates within a metabolic network that includes CPT1, CPT2, acyl-CoA synthetase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase.
In the context of the VHL-null 786-O cell line, disruption of ECI2 provides a valuable tool to dissect the role of fatty acid oxidation in ccRCC metabolic reprogramming. ccRCC tumors are characterized by lipid accumulation and reliance on fatty acid metabolism for energy and lipid biosynthesis. The ECI2 knockout model allows researchers to examine how loss of unsaturated fatty acid oxidation affects cellular lipid homeostasis, mitochondrial respiration, and redox balance under the pseudohypoxic conditions driven by HIF-2??. This model is particularly suited for investigating the crosstalk between HIF-2???Cmediated signaling and peroxisomal function, as well as the impact on ATP generation and overall tumor cell fitness.
This polyclonal knockout cell population is optimized for a range of functional analyses, including western blotting and RT-qPCR to confirm ECI2 inactivation, metabolic flux analysis using Seahorse XF analyzers or radiolabeled fatty acid oxidation assays (e.g., 14C-palmitate), and metabolomic profiling by LC-MS. The cells can be used in lipid droplet staining with Oil Red O, ATP measurements, ROS detection, and cell proliferation assays to assess metabolic phenotypes. These applications support studies on lipid metabolism in ccRCC, identification of synthetic lethal interactions with VHL deficiency, and evaluation of ECI2 as a potential therapeutic target in peroxisomal disorders and metabolic syndrome. For additional technical information, please contact Ascent Research.
