Human VHL Knockout Cell Line-HEK293T
Cat.No. : CSC-RT2714
Host Cell: HEK293T Target Gene: VHL
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2714 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human VHL using CRISPR/Cas9. |
| Target Gene | VHL |
| Host Cell | HEK293T |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid nirtogen |
| Species | 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. |
| 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 1:3-1:6. |
| 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
The VHL (von Hippel-Lindau) gene is a key component in regulating cellular functions related to oxygen sensing and tumor suppression. The VHL gene is located on the short arm of chromosome 3 (3p25-26) and produces the VHL protein, which plays an important role in controlling cell growth, forming blood vessels, and regulating other important cellular processes. One of the most well-characterized functions of the VHL protein is its role in degrading hypoxia-inducible factor (HIF). HIF is a transcription factor activated under low oxygen conditions (hypoxia) to promote the expression of genes involved in angiogenesis, metabolism, and survival. Under normoxic conditions, the VHL protein ubiquitinates HIF, leading to its degradation and preventing inappropriate gene activation.
Mutations or deletions in the VHL gene can adversely affect the function of the VHL protein, leading to the development of various diseases, most notably Hippel-Lindau disease. This rare genetic disease is characterized by the formation of tumors and cysts in multiple organs, including the brain, kidneys, pancreas, adrenal glands, and retina. Depending on the type and location of these tumors, affected individuals may experience a range of symptoms, from headaches and vision problems to high blood pressure and abdominal pain. In addition to Hippel-Lindau disease, mutations in the VHL gene have been associated with an increased risk of other cancers, particularly clear cell renal cell carcinoma (ccRCC) and pheochromocytoma. Research into the VHL gene and its function has paved the way for deeper insights into cancer biology and potential treatments.
The Wnt/β-catenin signaling pathway plays a critical role in development and adult tissue homeostasis by controlling cell proliferation and cell fate determination. In this pathway, the transcription factors TCF/LEF are key components that repress target gene expression by recruiting corepressors or activate target gene expression by recruiting β-catenin in the absence or presence of Wnt signals. Here, researchers show that the von Hippel-Lindau protein (pVHL) controls the stability of TCF/LEF proteins as substrate recognition components of the E3 ubiquitin ligase complex. Unexpectedly, pVHL directly binds to TCF/LEF and promotes their proteasomal degradation independent of E3 ubiquitin ligase activity. Knockout of vhl in zebrafish embryos leads to a reduction in dorsal habenular neurons, and this effect is upstream of the dorsal habenular neuron phenotype in tcf7l2-null mutants. These studies reveal a previously unknown mechanism for protein stability regulation of TCF/LEF transcription factors and demonstrate that pVHL contains a 26S proteasome-binding domain that drives ubiquitin-independent proteasomal degradation.
Here, researchers used CRISPR/Cas9-mediated gene editing to generate VHL knockout HEK293T cells (Figure 1A). The generated VHL knockout line has a premature stop codon at exon 1, which results in the depletion of both isoforms (Figure 1C). As expected, knockout of pVHL enhanced basal Wnt reporter activity (Figure 1C). HEK293T cells were considered a Wnt shut-off cell line. Addition of the GSK3 inhibitor 6-bromoindirubin-3'-oxime (BIO) to HEK293T cells induced Wnt reporter activity (Figure 1D). To further investigate the effect of VHL depletion on Wnt activity, VHL-depleted HEK293T cells were treated with BIO, and Wnt reporter activity was measured. In the context of BIO treatment, knockout of pVHL further increased Wnt reporter activity (Figure 1D). Taken together, these results suggest that pVHL inhibits Wnt/β-catenin signaling.
In addition, pVHL knockout significantly increased HIF-1α protein levels. Similarly, pVHL knockout significantly increased TCF7, TCF7L1, and TCF7L2 protein levels (Figure 1K). Quantitative real-time RT-PCR analysis showed that knockout of pVHL did not alter the mRNA levels of TCF7, TCF7L1, or TCF7L2 (Figure 1L). Moreover, reintroduction of pVHL into VHL knockout HEK293T cells neutralized this effect, as TCF7, TCF7L1, TCF7L2, and HIF-1α protein levels were significantly reduced (Figure 1M). To verify this result, the researchers further reintroduced pVHL into VHL-deficient ccRCC 786-O cells. Reintroduction of pVHL resulted in a decrease in endogenous TCF7 and TCF7L2 protein levels ( Figure 1N ). These results indicate that VHL knockout is specific and that pVHL promotes TCF/LEF protein degradation in vitro.
Figure 1. pVHL inhibits Wnt/β-catenin signaling and stabilizes TCF/LEF protein. (Wang C, et al., 2021)
The Human VHL Knockout Cell Line - HEK293T is a powerful tool for biomedical research, especially in the fields of oncology, genetics, and cell biology. Here are the main applications of this cell line:
Cancer Research: The VHL gene plays a key role in the regulation of hypoxia-inducible factor (HIF). Knockout of VHL in HEK293T cells provides a model for studying tumorigenesis and cancer progression, especially for cancers such as renal cell carcinoma where VHL mutations are prevalent. Researchers can study the mechanisms by which loss of VHL function leads to uncontrolled cell proliferation and tumor growth.
Drug Screening: By using VHL knockout cells, researchers can evaluate the efficacy of drugs that target the HIF pathway or other pathways that are dysregulated by the loss of VHL. This can ultimately facilitate the development of novel cancer treatments.
Hypoxia Research: VHL normally regulates the degradation of HIF, and its loss can mimic a hypoxic environment. Researchers use these cells to understand how hypoxia-inducible factors affect gene expression, cell metabolism, and survival under low oxygen conditions.
Pathway Analysis: This cell line provides a system to dissect signaling pathways and downstream effects of VHL loss. Researchers can analyze changes in cellular signaling networks and identify key drivers of altered cellular behavior as a result of VHL knockout.
Biomarker Discovery: Utilizing VHL knockout HEK293T cells can aid in the discovery of new biomarkers for cancer and other diseases. By comparing these cells to wild-type controls, researchers can identify specific proteins, RNAs, or metabolites that are differentially expressed as a result of VHL loss. These biomarkers can be used for diagnostic purposes or as therapeutic targets.
For research use only. Not intended for any clinical use.
