The DNM1L Knockout HEK293T Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population in which the DNM1L gene, encoding the dynamin-related GTPase DRP1, has been disrupted. This loss-of-function model provides a genetically defined system for dissecting mitochondrial and peroxisomal fission mechanisms without clonal artifacts. The polyclonal format preserves heterogeneous editing events, offering a reproducible platform for functional studies of organelle dynamics, apoptosis, and metabolic regulation in a widely used human cell background.
HEK293T cells are human embryonic kidney epithelial cells that stably express the SV40 large T antigen, enabling episomal replication of SV40 origin-containing plasmids. This feature, combined with high transfectability and robust protein expression capacity, makes HEK293T a preferred host for exogenous gene expression, lentiviral and retroviral packaging, and CRISPR-based genome engineering. The cells’ epithelial origin and ease of manipulation facilitate detailed cell biological analyses, including live-cell imaging and biochemical fractionation, making them an ideal chassis for investigating mitochondrial network dynamics upon DNM1L disruption.
DNM1L/DRP1 is a cytosolic GTPase that translocates to the mitochondrial outer membrane in response to cellular cues, where it is recruited by adaptors MFF, MIEF1 (MiD49), and MIEF2 (MiD51), and cooperates with Fis1 to oligomerize into helical filaments that constrict and sever the organelle. Its activity is tightly regulated by phosphorylation through cyclin-dependent kinases (CDK1, CDK5), AMPK, PKA, CaMKII, and ERK1/2. Downstream of DRP1-mediated fission, mitochondrial fragmentation facilitates cytochrome c release and activation of caspase-9 and caspase-3, linking organelle shape to apoptosis. DRP1 also interacts with GDAP1, Bax, Bak, and OPA1-containing complexes, underscoring its central role in mitochondrial quality control and cell death pathways.
In the HEK293T background, knockout of DNM1L leads to a striking hyperfused mitochondrial reticulum, impaired mitophagy flux, and resistance to intrinsic apoptosis, mirroring phenotypes observed in primary cells and disease models. This polyclonal knockout population enables robust investigation of fission-dependent processes, including mtDNA distribution, mitochondrial calcium handling, and metabolic reprogramming, without confounding clonal selection effects. The model is particularly valuable for studying how upstream regulatory kinases and adaptor proteins modulate DRP1 localization and activity in a tractable epithelial system.
Representative applications include immunofluorescence-based mitochondrial morphology scoring, Seahorse metabolic flux analysis to assess oxidative phosphorylation and glycolysis, caspase-3/7 activation assays for apoptosis profiling, and mitophagy flux measurements using pH-sensitive reporters. The knockout cells are also suitable for drug screening targeting mitochondrial disorders and for co-expression studies with fluorescent organelle markers. For additional technical specifications, protocols, or custom services, please contact Ascent Research.
