Cat.No. : CSC-RT0067
Host Cell: HCT116 Target Gene: DNMT3B
Size: >1x10^6 cells/vial, 1 mL Validation: Sequencing
| Cat. No. | CSC-RT0067 |
| Description | 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 |
| Host Cell Species | Homo sapiens (Human) |
| Cell Type | Epithelial |
| Size | >1x10^6 cells/vial, 1 mL |
| SequencingResult | 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 25cm2 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 |
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)
A: DMEM supplemented with 10% fetal bovine serum.
It is not required to add the selection antibiotics when culturing the KO cells.
A: The knockout cell product is validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. Please find the detailed mutation info in the datasheet.
A: Single clonal cell.
A: No. This knockout cell product is generated using the CRISPR/Cas9 system to induce small insertions or deletions (indels) resulting in frameshift mutations. Although these frameshift mutations typically disrupt the coding gene, there is a possibility that the non-functional transcript may still be transcribed. Consequently, this could potentially yield misleading results when analyzed by RT-qPCR.
A: The cell line should be stored in liquid nitrogen for long-term preservation.
A: For most cases, we often keep at least 2 clones with different frameshift mutations. Please feel free to contact us to check if there are additional available clones.
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DNMT3B knockout cells lack the DNMT3B gene's expression and, therefore, have reduced DNA methylation activity. This cell line is very helpful for my research.
We are investigating the use of DNMT3B knockout cells in breast cancer therapy. Good experimental results were obtained. I recommend it.
I have used several gene knockout reagents in the past, but Human DNMT3B Knockout Cell Line-HCT116 is the best by far. It is very easy to use and produces reliable results.
The knockdown efficiency of Human DNMT3B Knockout Cell Line-HCT116 is simply amazing. It has made my experiments much more efficient and streamlined.
I am so glad that I discovered Human DNMT3B Knockout Cell Line-HCT116. It has made my research much more productive, and the results are always consistent.
I cannot recommend Human DNMT3B Knockout Cell Line-HCT116 enough. It is incredibly effective and has made a huge difference in my research.
Human DNMT3B Knockout Cell Line-HCT116 has made gene editing so much simpler. It is easy to use and consistently produces great results.
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