Cat.No. : CSC-RT0039
Host Cell: HCT116 Target Gene: AKT2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT0039 |
| Description | HCT116-AKT2 (-/-) is a cell line with a homozygous knockout of human AKT2 |
| Target Gene | AKT2 |
| 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 |
Homologous recombination repair (HRR), nonhomologous end joining (NHEJ), and alternative NHEJ are the main pathways for cellular processing of DNA double-strand breaks (DNA-DSBs). Their functions play an important role in the radioresistance of tumor cells. There are conflicting data regarding the role of Akt in homologous recombination (HR), namely the regulation of Rad51 as a major protein of this pathway. This study was designed to investigate the specific involvement of Akt isoforms in HRR. HCT116 colon cancer cells with stable AKT knockout and siRNA-mediated AKT knockdown phenotypes were used to investigate the role of Akt1 and Akt2 isoforms in HR. The results clearly showed that AKT1 Knockout cell-HCT116 and AKT2 Knockout cell-HCT116 showed a significant reduction in Rad51 foci formation at 6 h after irradiation compared to parental cells. Depletion of Akt1 and Akt2 protein levels and inhibition of Akt kinase activity resulted in an increase in the amount of residual γH2AX in CENP-F-positive cells, which mainly represent cells in the S and G2 phases. In addition, inhibition of NHEJ and HR using DNA-PK and Rad51 antagonists resulted in enhanced radiosensitivity of AKT1 and AKT2 knockout cells compared to wild-type cells. Together, these data suggest that both Akt1 and Akt2 are involved in repairing DSBs via HRR.
The number of Rad51 foci in HCT116 AKT1 and AKT2 knockout cells was significantly suppressed 6 h after exposure to irradiation IR compared to parental HCT116 control cells. Interestingly, however, treatment with the Akt inhibitor MK2206 for 2 h before radiation exposure resulted in only a slight but non-significant reduction in Rad51 foci 6 h after IR (Figure 1C). To evaluate whether a non-functional NHEJ pathway could increase Rad51 foci formation after radiation exposure, siRNA-mediated double knockdown of AKT1 and AKT2 was performed in HCT116 DNA-PK-deficient cells. As shown in Figure 1D, in the absence of functional cNHEJ repair, the number of Rad51 foci increased by approximately 20% compared to cNHEJ-proficient cells. This was due to compensatory stimulation of HR. This idea was supported by Western blot data showing that NHEJ-deficient cells presented upregulated Rad51 protein expression (Figure 1E). siRNA-mediated downregulation of both Akt isoforms resulted in a significant reduction in Rad51 focus formation in NHEJ-proficient and deficient cells (Figure 1D). These data clearly indicate that Akt depletion significantly impairs Rad51 loading of DNA-DSBs, leading to impaired HR repair processes.
Figure 1. Determination of Rad51 foci formation and protein levels in HCT116 parental and AKT1/AKT2 knockout cells. (Mohammadian Gol T, et al., 2019)
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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Using the AKT2 knockout HCT116 cells, we have been able to scrutinize cell survival and apoptosis mechanisms more effectively. The clarity and consistency of the results have tremendously improved the accuracy of our experimental interpretations.
The Human AKT2 Knockout Cell Line-HCT116 has significantly advanced our cancer research. The complete suppression of the AKT2 gene has provided invaluable insights into signal transduction pathways and tumor growth, helping us to identify potential therapeutic targets.
The quality and performance of the Human PTEN Knockout Cell Line-HCT116 have been outstanding. It has been instrumental for our drug screening assays and mechanistic studies.
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