KMT2C Knockout HAP1 Cell Line

The choice depends on whether you are studying KMT2C (MLL3, ALR)'s role as a H3K4 monomethyltransferase at enhancers or modeling KMT2C-mutation-associated cancers and Kleefstra syndrome 2. The Knockout line is the standard tool for asking whether KMT2C is required for these processes — KMT2C is the catalytic subunit of the COMPASS-like complex (with PTIP, NCOA6, PA1, UTX/KDM6A, ASH2L, RBBP5, WDR5, DPY30) that deposits H3K4me1, the principal enhancer mark. Overexpression is useful for studying KMT2C in heterologous expression contexts. For cancer epigenetics research, the EDITGENE KMT2C Knockout in HAP1 is highly informative — KMT2C is one of the most frequently mutated genes in human cancers (loss-of-function in breast, bladder, lung, and many other cancers), functioning as a tumor suppressor. Rescue with wild-type or catalytically-dead KMT2C is the standard specificity control. The knockout is valuable for studying enhancer biology, KMT2C-mutant cancer dependencies, and emerging epigenetic therapy approaches — KMT2C-mutant cancers may show synthetic lethality with KDM5 (H3K4me-erasers) inhibition.

Primary applications: • H3K4me1 enhancer mark: H3K4me1 ChIP-seq analysis to characterize KMT2C-dependent enhancer methylation. • KMT2C-mutant cancer dependencies: KDM5 inhibitor sensitivity studies given the proposed KMT2C-loss / KDM5-inhibition synthetic lethality. • COMPASS-like complex: co-immunoprecipitation of KMT2C complex components (PTIP, UTX/KDM6A, ASH2L) in the absence of KMT2C. • Enhancer biology: H3K4me1 and enhancer activity (H3K27ac, enhancer RNA) analysis at KMT2C target enhancers. EDITGENE recommends this model for researchers investigating enhancer biology, COMPASS-like complex function, and KMT2C-mutant cancer therapeutic strategies.

Yes, with considerations for KMT2C's large size: • Construct design: KMT2C encodes a very large protein (~5,000 aa) — full-length cDNA (~14 kb) rescue is technically challenging. Use codon-modified KMT2C with a small C-terminal tag (FLAG, HA); preserve SET domain (H3K4 methyltransferase activity), PHD fingers (chromatin reader), and FYRN/FYRC motifs. • Catalytically-dead rescue: SET domain catalytic residue mutations abolish methyltransferase activity and serve as the standard specificity control. • Domain-focused rescue: SET-containing C-terminal fragments are sometimes used when full-length rescue is impractical — these can be functional for enhancer methylation. • Functional readout: rescue should restore H3K4me1 levels at KMT2C target enhancers measured by ChIP-seq. HAP1-specific considerations: • Diploidization: HAP1 cells gradually diploidize during extended culture — confirm ploidy by flow cytometry at the time of phenotypic assay. • Integration site sensitivity: position effects on transgene expression are more pronounced in near-haploid backgrounds; generating multiple independent rescue clones is strongly recommended. • Transduction efficiency: HAP1 transduces with lentivirus at moderate efficiency — increase MOI compared to standard immortalized lines.