The ACSL4 Knockout MDCK Cell Line is a CRISPR/Cas9-edited knockout cell line derived from Madin-Darby Canine Kidney (MDCK) epithelial cells, designed for the disruption of the ACSL4 gene. This loss-of-function model enables investigation of ACSL4-dependent processes in an immortalized canine kidney epithelial background. The cell line is generated using CRISPR/Cas9-mediated gene disruption, resulting in a stable knockout of the ACSL4 locus without introducing exogenous sequences. It serves as a defined genetic tool for dissecting the role of ACSL4 in lipid metabolism, ferroptosis regulation, and epithelial cell physiology.
The parental MDCK cell line is a widely used model for polarized epithelial monolayers, originally derived from the distal tubule/collecting duct of canine kidney. MDCK cells form tight junctions, develop apical-basal polarity, and exhibit vectorial transport, making them a gold standard for studying epithelial barrier function, protein trafficking, and ion transport. Their well-characterized differentiation and robust growth in two- and three-dimensional cultures provide a reliable platform for evaluating gene knockout effects on epithelial architecture and signaling.
ACSL4 (acyl-CoA synthetase long-chain family member 4) preferentially activates long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic and eicosapentaenoic acid, converting them into acyl-CoA thioesters. These PUFA-CoAs are substrates for phospholipid remodeling, enriching cellular membranes with oxidizable phospholipids that sensitize cells to ferroptosis, an iron-dependent form of regulated necrosis driven by lipid peroxidation. ACSL4 is positively regulated by STAT3, PPAR??, and LXR, and its expression is modulated by insulin and inflammatory cytokines like TNF-??. The resulting PUFA-CoA esters are channeled into downstream pathways including eicosanoid production, lipid droplet biogenesis, and phospholipid synthesis. In the ferroptosis cascade, ACSL4 acts upstream of lipoxygenases such as ALOX5 and ALOX15, which oxidize PUFA-containing phospholipids, generating lipid peroxides. This pro-ferroptotic activity is counterbalanced by the glutathione peroxidase GPX4, which reduces phospholipid hydroperoxides. Thus, the interplay among ACSL4, ALOX15/ALOX5, and GPX4 determines cellular sensitivity to ferroptosis induction by agents like erastin and RSL3.
In MDCK cells, ACSL4 knockout is particularly relevant for dissecting ferroptosis in renal epithelium, as kidney tissue is highly susceptible to ferroptotic damage during ischemia-reperfusion injury and nephrotoxicity. Disruption of ACSL4 in this model allows researchers to explore how lipid metabolism intersects with epithelial polarity, barrier function, and cell death pathways. Given that MDCK cells express key transporters and maintain differentiated epithelial characteristics, the knockout line facilitates studies on how ACSL4 influences lipid droplet formation, prostaglandin secretion, and membrane phospholipid composition in a polarized context. Additionally, it offers a tool to evaluate the contribution of ACSL4 to tumorigenic properties when MDCK cells are transformed or used in cancer-relevant assays.
Researchers can employ this cell line in a variety of assays, including Western blotting and RT-qPCR to confirm ACSL4 ablation, lipidomics profiling to assess changes in phospholipid species, and ferroptosis induction experiments using erastin or RSL3 followed by cell viability or ROS measurements. Functional studies may involve the C11-BODIPY lipid peroxidation sensor, immunofluorescence for lipid droplet or tight junction markers, and transepithelial electrical resistance (TEER) assays to monitor barrier integrity. The ACSL4 Knockout MDCK Cell Line is thus ideal for investigating ferroptosis mechanisms, lipid metabolism, and kidney epithelial biology. For additional information or technical support, please contact Ascent Research.
