Human HIF1A Knockout Cell Line-Hela
Cat.No. : CSC-RT0517
Host Cell: Hela Target Gene: HIF1A
Size: >1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0517 |
| Cell Line Information | This cell line is a stable cell line with a homozygous knockout of human HIF1A using CRISPR/Cas9. |
| Target Gene | HIF1A |
| Gene ID | 3091 |
| Genotype | HIF1A (-/-) |
| Host Cell | Hela |
| Cell Type | Epithelial |
| Size | >1x10^6 cells/vial, 1mL |
| Sequencing Result | Homozygous knockout: 1 bp insertion in exon |
| 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 |
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Background
Case Study
Applications
Hypoxia-inducible factor 1-α (HIF1A) is a key transcription factor that plays a crucial role in the cellular response to low oxygen levels (hypoxia). Under normoxic conditions (normal oxygen levels), HIF1A is hydroxylated by prolyl hydroxylases, which ultimately target it for rapid ubiquitination and proteasomal degradation. This process is mediated by the von Hippel-Lindau (VHL) tumor suppressor protein, which recognizes hydroxylated HIF1A and marks it for destruction. Therefore, under normoxic conditions, HIF1A levels remain low.
When oxygen levels drop, prolyl hydroxylase activity decreases, leading to HIF1A stabilization and accumulation. Once stabilized, HIF1A translocates to the nucleus, where it dimerizes with HIF-1β. The HIF-1 complex then binds to hypoxia response elements (HREs) in the promoter regions of various target genes, activating transcription of genes involved in the adaptive response to hypoxia. These genes are involved in processes such as angiogenesis, erythropoiesis, glucose metabolism, and cell survival, which together help organisms cope with low oxygen levels.
HIF1A has broad implications in health and disease. Its role in promoting angiogenesis is particularly important in cancer, as tumor cells often exist in hypoxic environments. By enabling cancer cells to adapt and survive in low oxygen conditions, HIF1A contributes to tumor growth and metastasis. Therefore, it has become an important target for therapeutic intervention in tumors. In addition, HIF1A has been implicated in ischemic diseases such as stroke and myocardial infarction, in which tissue oxygen supply is compromised. Therefore, understanding the molecular mechanisms that regulate HIF1A could provide insights into developing treatments for a variety of hypoxia-related diseases.
Hypoxia-inducible factor 1 (HIF-1) is a key transcriptional mediator of the cellular response to hypoxia and is also involved in cancer progression. Regulation of its oxygen-sensitive HIF-1α subunit involves post-translational modifications that control its stability, subcellular localization, and activity. Phosphorylation of the HIF-1α C-terminal domain by ERK1/2 promotes nuclear accumulation of HIF-1α and stimulates HIF-1 activity, whereas lack of this modification triggers nuclear export of HIF-1α and its association with mitochondria. On the other hand, modification of the N-terminal domain of HIF-1α by CK1δ attenuates HIF-1 activity by impeding the formation of HIF-1α/ARNT heterodimers.
To investigate the interplay between the two antagonistic HIF-1α phosphorylations by CK1δ and ERK1/2 and their role in HIF-1α subcellular distribution and activity, the researchers constructed a number of HIF-1α mutant forms that combined phospho-deficient and phospho-mimetic mutations at two sites of the two kinases and expressed them in HIF1A knockout HeLa cell lines. In addition, they investigated the interactome of non-nuclear HIF-1α and how it might be affected by CK1δ. The study found that modification of non-nuclear HIF-1α by CK1δ leads to its release from mitochondria and its binding to microtubules through interactions between the N-terminal part of HIF-1α and tubulin. Furthermore, these results suggest that CK1δ-stimulated binding of endogenous HIF-1α to microtubules is most pronounced during mitosis and is required for the symmetric delivery of HIF-1α to daughter nuclei during cell division.
Figure 1. CK1δ phosphomimic mutations reduce mitochondrial association of non-nuclear HIF-1α. A Fluorescence microscopy images of HIF1A knockout HeLa cells expressing GFP-HIF-1α wt-SA or GFP-HIF-1α SD-SA grown under hypoxia (1% O2). B Western blot analysis of proteins recovered from soluble and microtubule-enriched fractions of HIF1A knockout HeLa cells expressing GFP-HIF-1α SD-SA under hypoxia (1% O2) using antibodies against the indicated proteins. (Arseni, Christina, et al. 2024)
Hypoxia Research: HIF1A (hypoxia inducible factor 1-alpha) plays a key role in the cellular response to low oxygen conditions. By using a HIF1A knockout cell line, researchers can study specific pathways and genes regulated by HIF1A under hypoxic conditions, which is critical to understanding diseases such as cancer where hypoxia is a common feature.
Cancer Research: HIF1A is closely associated with tumor progression, angiogenesis, and metastasis. Using the HIF1A knockout HeLa cell line, it is possible to study how the loss of this gene affects cancer cell behavior, providing insights that may lead to new therapeutic targets.
Gene Function Analysis: By observing the phenotypic consequences of deleting HIF1A, researchers can better understand the role of this gene in various cellular processes. This contributes to a broader understanding of gene regulation and function in human cells.
Drug Screening and Development: This cell line serves as a valuable tool for testing the efficacy and mechanism of new drugs targeting the HIF1A-regulated hypoxia pathways. It allows for comparison of drug effects in the presence and absence of HIF1A.
Metabolic Research: HIF1A affects key metabolic pathways, and its knockout helps to elucidate its role in cellular metabolism. Studying these pathways may reveal how cells adjust their metabolism in response to different environmental conditions.
Differentiation and Developmental Biology: HIF1A is involved in various developmental processes. The HIF1A knockout HeLa cell line can be used to study how the loss of HIF1A affects cell differentiation and development-related pathways.
For research use only. Not intended for any clinical use.
