DNMT3B Knockout HaCaT Cell Line

The DNMT3B Knockout HaCaT Cell Line is a CRISPR/Cas9-edited knockout cell line featuring targeted disruption of the DNMT3B gene in the human HaCaT keratinocyte background. This loss-of-function model is designed to eliminate de novo DNA methyltransferase activity encoded by DNMT3B, enabling precise investigation of DNA methylation-dependent gene silencing in a well-characterized epithelial system. The cell line is produced without claims of clonality, providing a representative pool of edited cells suitable for population-level epigenetic and functional studies.

HaCaT cells are a spontaneously immortalized, non-tumorigenic human keratinocyte line isolated from adult skin and widely adopted as a model for normal epidermal keratinocytes. These cells retain the capacity for epidermal barrier formation, participate in wound healing processes, and secrete a range of cytokines, making them highly relevant for studying skin biology. Their stable, near-diploid karyotype and robust in vitro growth characteristics facilitate reproducible experimental conditions for gene-editing studies.

DNMT3B is a de novo DNA methyltransferase that establishes CpG methylation patterns, critical for gene silencing, genomic imprinting, and repression of repetitive elements. It is regulated by transcription factors SP1 and SP3, the polycomb protein EZH2, miR-29 family members, and the RAS/MEK/ERK pathway. DNMT3B interacts with DNMT3L, UHRF1, HDAC1, HP1, and NuRD complex components to coordinate epigenetic silencing. Downstream targets silenced by DNMT3B include CDKN2A (p16INK4a), CDH1 (E-cadherin), MLH1, retrotransposons (LINE-1, Alu), and imprinted genes (H19, IGF2). The methylation reaction utilizes SAM and operates within a network involving DNMT1, DNMT3A, MBD proteins, and SETDB1. Knockout of DNMT3B causes global hypomethylation, reactivation of tumor suppressors and transposable elements, and may impair genomic stability.

In the HaCaT keratinocyte context, DNMT3B knockout provides a powerful tool to dissect the role of DNA methylation in epidermal homeostasis. Loss of DNMT3B is anticipated to impair proper differentiation, alter wound healing responses, and compromise genomic stability by derepressing repetitive elements. Reactivation of tumor suppressors such as CDKN2A and CDH1 may affect cell cycle progression and adhesion, offering insight into early epigenetic events in skin carcinogenesis. This model allows researchers to directly assess how DNMT3B-dependent methylation influences keratinocyte behavior under various stimuli.

This knockout cell line supports diverse applications including epigenetic study of keratinocytes, cancer epigenetics, DNMT inhibitor screening, wound healing assays, and skin differentiation models. Analytical methods such as bisulfite sequencing, RT-qPCR, MeDIP-seq, and immunofluorescence for 5-methylcytosine can be applied. Functional readouts including scratch wound migration, proliferation, and apoptosis assays are facilitated. For additional product information, please contact Ascent Research.