Human DNMT3B Knockout Cell Line-HCT116
Cat.No. : CSC-RT0067
Host Cell: HCT116 Target Gene: DNMT3B
Size: >1x10^6 cells/vial, 1 mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0067 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human DNMT3B using CRISPR/Cas9. |
| Target Gene | DNMT3B |
| Gene ID | 1789 |
| Genotype | DNMT3B (-/-) |
| Host Cell | HCT116 |
| Cell Type | Epithelial |
| Size | >1x10^6 cells/vial, 1 mL |
| Sequencing Result | Homozygous: 46 bp deletion in exon |
| Culture Properties | Adherent |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Media Type | Cells were cultured in DMEM supplemented with 10% fetal bovine serum. |
| Growth Properties | Cells are cultured as a monolayer at 37°C in a humidified atmosphere with 5% CO2. Split at 80-90% confluence, approximately 1:4-1:6. |
| Freeze Medium | Complete medium supplemented with 10% (v/v) DMSO |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
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Background
Case Study
Applications
DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) is a key enzyme involved in the process of DNA methylation, a critical epigenetic modification that regulates gene expression, development, and genome integrity. DNMT3B is unique in its role during early embryogenesis and de novo methylation, meaning that it establishes new DNA methylation patterns on previously unmethylated cytosine residues. This enzymatic activity is essential for normal development, genomic imprinting, and X chromosome inactivation in mammals. Unlike DNMT1, which primarily maintains methylation during DNA replication, DNMT3B targets both symmetric CpG sites and non-CpG environments, increasing the complexity and specificity of epigenetic regulation.
Mutations in the DNMT3B gene have been associated with various diseases, most notably immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome. ICF syndrome is a rare autosomal recessive disorder characterized by immune deficiency, facial anomalies, and chromosomal instability, which emphasizes the important role of this enzyme in maintaining genomic stability and normal physiological function. In addition to its developmental role, DNMT3B has also been implicated in cancer. Aberrant expression and mutations of DNMT3B are frequently observed in various malignancies, where they lead to dysregulated gene expression and thus tumorigenesis. This makes DNMT3B both a potential biomarker for cancer prognosis and a target for epigenetic therapy.
Mouse embryonic fibroblasts derived from DNMT3b knockout embryos exhibit DNA damage and chromosomal instability, suggesting that DNMT3b plays a key role in genomic stability. Loss-of-function mutations in DNMT3b are specifically seen in a rare human genetic disorder, immunodeficiency-centromere instability-facial dysmorphism (ICF type 1) syndrome. Studies have shown that DNMT3b is recruited to GC-rich (pericentromeric) regions to maintain chromosomal stability through interaction with the centromeric protein CENP-C. Thus, the major genomic regions affected by DNMT3b loss-of-function in ICFs are pericentromere noncoding repetitive elements, where GC regions are hypomethylated, coinciding with the centromeric DNA breaks observed in ICF cells. However, how DNMT3b dysfunction increases DNA damage and centromeric instability remains an open question.
In this study, the researchers used human DNMT3B Knockout Cell Line-HCT116, in which both alleles of DNMT3b were interrupted by homologous recombination, and ICF cells carrying a DNMT3b loss-of-function mutation to address this question. Analysis revealed that R-loops are responsible for the significant DNA damage signature observed in DNMT3b knockout in HCT116 (BKO) and DNMT3b loss-of-function mutations in ICF lymphocytes. Sites of DNA damage in BKO cells were mapped to repetitive satellite sequences and rDNA genes. In BKO and ICF cells, (peri)centromeric R-loops are cleaved and removed by the endonucleases XPG and XPF. Depletion of XPG and XPF resulted in an increase in R-loops and a decrease in γH2AX associated with (peri)centromeric DNA sequences in both BKO and ICF cells. DNMT3b dysfunction significantly increased the sensitivity of R-loops to the cleavage process. Finally, the study showed that DNA double-strand breaks (DSBs) at centromeres may be repaired via an error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunction compromises centromere integrity via R-loop-mediated DNA damage and repair.
Figure 1.R-loops are the sources of DNA damage in DNMT3B Knockout Cell Line-HCT116 (BKO) and ICF cells. (Shih, Hsueh-Tzu, et al. 2022)
The DNMT3B gene encodes DNA methyltransferase 3β, an enzyme that is essential for de novo DNA methylation and regulation of gene expression. DNMT3B knockout cell line-HCT116 has become valuable tools for a variety of research applications. Here are some important applications:
Epigenetic Research: DNMT3B knockout cell lines facilitate the study of DNA methylation patterns and how they affect gene expression, epigenetic modifications, and cellular processes such as differentiation and development.
Cancer Research: DNMT3B knockout HCT116 cells facilitate the understanding of the role of DNMT3B in tumorigenesis. Researchers use these cells to explore how changes in DNA methylation contribute to cancer initiation, progression, and resistance to therapy.
Genetic Disease Research: This cell line is used to study genetic diseases associated with abnormal DNA methylation, such as ICF (immunodeficiency, centromere instability, and facial anomalies) syndrome, which is associated with DNMT3B mutations.
Drug Development: DNMT3B knockout HCT116 cells can be used to screen and develop drugs that target the DNA methylation pathway. These cells can help identify compounds that may modulate DNA methylation for therapeutic effects.
Developmental Biology: This cell line can serve as a model to study the effects of DNMT3B loss during cell differentiation. This can help understand the role of DNA methylation during development and may have implications for regenerative medicine and stem cell research.
For research use only. Not intended for any clinical use.
