Cat.No. : CSC-RT2793
Host Cell: Hela Target Gene: ATF4
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2793 |
| Description | This cell is a stable cell line with a homozygous knockout of human ATF4 using CRISPR/Cas9. |
| Target Gene | ATF4 |
| Host Cell | Hela |
| Host Cell Species | Homo sapiens (Human) |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| 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 |
| 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 |
Mitochondrial respiration is essential for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors such as aspartate, an essential substrate for nucleotide synthesis. Here, researchers show that, in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. In the context of ETC inhibition, exogenous asparagine restores proliferation, ATF4 and mTORC1 activity, and mTORC1-dependent nucleotide synthesis, suggesting that asparagine relays active respiration to ATF4 and mTORC1. Finally, researchers show that the combination of the ETC inhibitor metformin (to limit tumor asparagine synthesis) with either asparaginase or dietary asparagine restriction (to limit tumor asparagine consumption) effectively inhibits tumor growth in multiple mouse cancer models.
The extent to which exogenous asparagine limited ATF4 activation by ETC inhibition correlated with the ability of asparagine to restore mTORC1 activity: no rescue of mTORC1 by asparagine was observed with antimycin A, where asparagine resulted in only partial rescue of ATF4, whereas in the presence of complex I and complex V inhibition, asparagine fully restored mTORC1 activity, whereas ATF4 activation was completely blocked (Figure 1A, B). Thus, the researchers proposed that ATF4 activation may contribute to the reduced mTORC1 activity resulting from respiratory impairment. To test this possibility, they engineered ATF4 knockout HeLa cells to constitutively express ASNS, thereby uncoupling ASNS activity and asparagine synthesis from ATF4 expression. These data indicate that in the absence of ATF4, mTORC1 activity is insensitive to ETC inhibition (constitutively active) (Figure 1E), raising the possibility that asparagine signals respiratory activity to mTORC1 at least in part through ATF4.
Figure 1. Asparagine relays mitochondrial respiration to ATF4 and mTORC1. (Krall A S, et al., 2021)
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.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
The high viability and robustness of the Human ATF4 Knockout HeLa cells were impressive. They adapted well to our standard lab conditions and showed consistent growth rates.
Using the ATF4 Knockout Cell Line - HeLa significantly reduced the time we needed to generate our own knockout models. This off-the-shelf solution accelerated our research timeline, allowing us to focus more on downstream applications and data analysis. Highly recommended for any lab in need of a quick and reliable knockout solution.
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