DNMT3A Knockout HCT 116 Polyclonal Cells consist of a CRISPR/Cas9-edited polyclonal population derived from the HCT 116 human colorectal carcinoma cell line, engineered for targeted disruption of the DNMT3A gene. This loss-of-function model enables systematic investigation of de novo DNA methylation and its regulatory roles in gene expression, genomic stability, and oncogenic signaling. By eliminating DNMT3A function, the cells provide a reproducible platform for dissecting epigenetic mechanisms and assessing therapeutic responses in a well-characterized mismatch repair-deficient background.
The HCT 116 host cells are epithelial in origin and widely used in colorectal cancer research due to their defined genetic features, including an MLH1-deficient mismatch repair defect and a KRAS G13D activating mutation. These characteristics confer a hypermutable phenotype and constitutively active RAS signaling, making HCT 116 a relevant model for studying tumorigenesis and drug resistance. The polyclonal knockout population retains the heterogeneity of the parental line while uniformly lacking DNMT3A protein expression, offering a robust tool for population-level epigenetic analyses without clonal artifacts.
DNMT3A encodes a DNA methyltransferase responsible for establishing and maintaining genomic cytosine methylation patterns, particularly at CpG dinucleotides, and is a key epigenetic silencer of differentiation-associated genes. It functions in complexes with DNMT3L and DNMT3B, and interacts with HDAC1, HDAC2, EZH2, SUZ12, and UHRF1 to coordinate transcriptionally repressive chromatin states. DNMT3A activity is regulated by upstream factors including PU.1 and RUNX1, and mediates downregulation of target loci such as HOXA, HOXB, and MEIS1 gene clusters. Knockout of DNMT3A disrupts this network, leading to global hypomethylation and derepression of silenced genes, with downstream effects on cell proliferation and sensitivity to hypomethylating agents like decitabine and azacitidine.
In the HCT 116 context, loss of DNMT3A interacts with the existing MLH1 deficiency and KRAS mutation to alter the epigenetic landscape and potentially modify tumor phenotypes. The model is particularly suited for studying cooperativity between genetic and epigenetic drivers of colorectal cancer, as well as for evaluating DNA methylation-targeted therapies. Because DNMT3A mutations are recurrent in clonal hematopoiesis and myeloid malignancies, this cellular platform also serves as a valuable surrogate for dissecting pathways linking DNA methylation to hematopoietic stem cell self-renewal and leukemogenesis.
Key experimental applications include functional epigenomics, drug sensitivity profiling, and mechanistic studies of gene silencing. Researchers can validate knockout efficiency via Western blotting and RT-qPCR, assess global DNA methylation changes through 5-mC ELISA or bisulfite sequencing, and perform transcriptomic analysis by RNA-seq to identify reactivated genes. Proliferation and colony formation assays, along with decitabine sensitivity testing, enable interrogation of growth phenotypes and therapeutic vulnerabilities. For further information on DNMT3A Knockout HCT 116 Polyclonal Cells, please contact Ascent Research.
