Cat.No. : CSC-RT0035
Host Cell: HCT116 Target Gene: CDK2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT0035 |
| Description | HCT116-CDK2 (-/-) is a cell line with a homozygous knockout of human CDK2 |
| Target Gene | CDK2 |
| Host Cell | HCT116 |
| Host Cell Species | Homo sapiens (Human) |
| 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. |
| 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 |
Although cyclin-dependent protein kinase 2 (Cdk2) controls the G1/S transition and promotes DNA replication, it is not essential for cell cycle progression due to redundancy with Cdk1. However, Cdk2 also has non-redundant functions that can be shown in certain genetic backgrounds, where it has been reported to promote the G2/M DNA damage response checkpoint in TP53 (p53)-deficient cancer cells. However, in p53-deficient cells subjected to DNA damage, Cdk2 is inactivated by the CDK inhibitor p21. Therefore, here it was investigated whether Cdk2 differentially affects the checkpoint response in p53-deficient and p53-deficient cell lines. The study shows that Cdk2 stimulates the ATR/Chk1 pathway independently of p53 status and is required for an efficient DNA replication checkpoint response. Cdk2 is not required for a sustained DNA damage response and G2 arrest. Conversely, elimination of Cdk2 delayed S/G2 progression after DNA damage and accelerated the appearance of early markers of cell cycle exit.
In this study, bleomycin induced strong phosphorylation of Chk1, Chk2, and p53, and accelerated p21 induction in Cdk2 knockout (Cdk2−/−) HCT-116 cells (Figure 1a). In addition, Cdk2 knockdown (KD) did not impair Chk1 and Chk2 phosphorylation in U2OS and HeLa cells. While the WT cell population eventually entered mitosis, Cdk2−/− cells remained stably arrested in the G2 phase (Figure 1b).
In WT and p53−/− cells, bleomycin induced a large accumulation of CycB1-Cdk1 and CycA-Cdk1, while the CycB1-Cdk1 complex failed to increase in Cdk2−/− cells arrested in G2 (Figure 1c, 24 hours). Furthermore, unlike WT cells where CycB1 associates with active Cdk1 dephosphorylated at T14, Y15 (arrow, isoform 1), in Cdk2−/− and p53−/− cells, CycB1 primarily associates with the hyperphosphorylated Cdk1 isoform (3), which is inactive (Figure 1c, arrows). The reduced phosphorylation of Cdk1 in these cells is likely due to Chk1-dependent inhibition of Cdc25 family phosphatases, which dephosphorylate and activate Cdk1. Thus, at 24 h, Chk1 expression and phosphorylation levels were higher in Cdk2−/− and p53−/− cells than in WT cells (Figure 1a). In addition, Chk1 phosphorylates and activates Wee1 kinase 24, which inhibits Cdk2, further stabilizing G2 arrest (Figure 1d). Interestingly, Wee1 was overexpressed in Cdk2−/− cells, which may contribute to the rapid inhibition of Cdk1 phosphorylation (Figure 1d).
Figure 1. Cdk2 is not required for Chk1 activation upon DNA damage by bleomycin, and its absence slows S/G2 progression. (Bačević K, et al., 2017)
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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This knockout cell is really efficient, after transfecting and extracting the protein, I ran a western blot the next day and it knocked down very cleanly, just no expression, better than the cell line I ordered before.
This cell line is not much but the result is obvious, which shows its high transfection efficiency, although it is a little different from the expensive brand but it can be compared to about 70%, so it is not a loss to get it.
I once forgot to add transfection enhancer to a transfection experiment and I thought the result would not be very good, but in fact there was not much difference between the transfections, only a little difference between some groups.
After using this brand of cell lines I immediately got some other laboratory supplies of this brand, some of them are already in use, the results are quite good and the response is also good.
I remembered this brand when I got a lot of consumables for functional experiments from a reagent vendor on sale, and I ordered cell lines directly from this brand this time.
The CDK2 Knockout Cell at Creative Biogene is an ideal model for studying the effects of CDK2 on cancer cell growth.
I am studying the effects of these inhibitors on the CDK2 knockout cell line to evaluate their potential as cancer treatments. This cell line is very helpful for my research.
Great! The CDK2 gene is removed, effectively eliminating the production of the CDK2 protein.
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