Cat.No. : CSC-RT1458
Host Cell: HeLa Target Gene: WRN
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT1458 |
| Description | A stable cell line with a homozygous knockout of human WRN using CRISPR/Cas9. |
| Target Gene | WRN |
| Host Cell | HeLa |
| Host Cell Species | Homo sapiens (Human) |
| Shipping | 10^6 cells/tube |
| Storage | Liquid nitrogen |
| 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 |
| Gene Name | WRN |
| Gene ID | 7486 |
| 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 |
Mutations in the Werner (WRN) RECQL helicase are associated with premature aging syndrome (Werner syndrome, WS) and susceptibility to multiple cancers. In patients with solid cancers, WRN RECQL helicase deficiency is associated with improved overall survival after treatment with TOP1 inhibitors, which stabilize pathological TOP1-DNA-covalent-complexes (TOP1cc) on the genome. However, the potential mechanisms of WRN in conferring chemoresistance to TOP1 inhibitors remain unexplored. Here, genome-wide transcriptome analysis of ~25,000 genes revealed robust activation of NF-κB-dependent prosurvival genes in response to TOP1cc. CRISPR-Cas9 knockout, shRNA silencing, and underexpression of WRN render multiple cancers hypersensitive to TOP1 inhibitors. Researchers demonstrate that WRN orchestrates TOP1cc repair through both proteasome-dependent and proteasome-independent processes, unleashing robust ssDNA generation. This in turn provides signaling for CHK1-mediated NF-κB activation through IκBα-degradation and nuclear localization of p65 protein. Interestingly, site-directed mutagenesis and rescue experiments revealed that neither the RECQL helicase nor the DNA exonuclease enzymatic activities of WRN (WRNE84A, WRNK577M, and WRNE84A-K577M) are required for TOP1cc removal, ssDNA generation, and NF-κB activation signaling.
WRN knockout (WRN-KO) cells (with empty vector) were defective in removing TOP1cc compared to WRN-WT (Figure 1d, e). Ectopic expression of WRNWT in WRN-KO cells triggered complete removal of TOP1cc. Unexpectedly, ectopic expression of WRN single and double mutants for both exonuclease and helicase functions also almost completely rescued the phenotype of WRN-KO cells by enabling TOP1cc repair (Figure 1d, e). To further validate the redundant role of WRN exonuclease activity in TOP1cc removal, researchers generated the WRNΔExo deletion mutant (Figure 1b), which lacks the exonuclease domain (1-230 aa). Ectopic expression of WRNWT and WRNΔExo almost completely rescued the phenotype of WRN-KO cells, as TOP1cc was removed by CPT treatment. Together, these results suggest that non-enzymatic functions of WRN regulate TOP1cc removal by nanomolar CPT.
Figure 1. Non-enzymatic role of WRN in TOP1cc removal. (Gupta, Pooja, 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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We are thrilled with the Human WRN Knockout Cell Line-HeLa we purchased. The cell line has provided us with consistent, reproducible results, significantly advancing our DNA repair and aging research.
Using the Human WRN Knockout Cell Line-HeLa has significantly enhanced our research productivity. The knockout model precisely aligns with our study objectives, helping us save valuable time and resources.
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