PARK2 Knockout Cell Line-HeLa
Cat.No. : CSC-RT1868
Host Cell: HeLa Target Gene: PARK2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1868 |
| Cell Line Information | HeLa -PARK2(-/-) is a stable cell line with a homozygous knockout of human PARK2 using CRISPR/Cas9. |
| Target Gene | PARK2 |
| Host Cell | HeLa |
| Shipping | 1 vial of knockout cell line |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene Symbol | PARK2 |
| Gene ID | 5071 |
| 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
The PARK2 gene, also known as parkin RBR E3 ubiquitin protein ligase or simply parkin, is a gene that plays a key role in maintaining cellular homeostasis and the proper functioning of the nervous system. Parkin is part of the cellular machinery responsible for protein degradation and specifically functions as an E3 ubiquitin-protein ligase. This enzyme tags damaged or misfolded proteins with a molecule called ubiquitin, marking them for degradation by the proteasome, a protein complex responsible for proteolysis. This process plays a crucial role in maintaining protein quality control within the cell, thereby preventing the accumulation of toxic protein aggregates that can interfere with normal cellular function.
Mutations in the PARK2 gene are associated with autosomal recessive juvenile Parkinson's disease (AR-JP), a form of Parkinson's disease that develops at a younger age than the more common late-onset Parkinson's disease. To date, more than 100 PARK2 mutations have been identified, including deletions, duplications, and point mutations. These mutations often result in the production of non-functional or truncated parkin proteins, which impair its ubiquitin ligase activity. This disruption leads to the accumulation of substrates that should have been degraded, causing neuronal cell death and the neurodegenerative features observed in Parkinson's disease.
The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on mitochondrial outer membrane proteins, triggering the recruitment of the autophagic machinery. Here, researchers describe the detection of a specific 500 kDa phospho-ubiquitin-enriched PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. The formation of this complex is dependent on the presence of PINK1, which is absent in mutant forms of PARK2 and thus prevents mitophagy. These results suggest that a functional signaling complex is essential for the progression of mitophagy. Visualization of the PARK2 signaling complex represents a new marker for this critical step in mitophagy and can be used to monitor the progression of mitophagy in PARK2 mutants and reveal additional upstream factors required for PARK2-mediated mitophagy signaling.
The results presented here demonstrate that formation of the PARK2 signaling complex is sensitive to PARK2 mutations and correlates with blockade of mitophagy, thus demonstrating its utility as a tool for analyzing signaling defects in PARK2 mutants (Figure 1A and B). To eliminate any interference by endogenous PARK2, knockout strains were created using CRISPR/Cas9, and elimination of PARK2 was confirmed by immunoblotting (Figure 1C) and sequencing. Recruitment of PARK2 to mitochondria was assessed by cell fractionation (Figure 1D). Both wild-type FLAG-PARK2 and the R275W mutant were efficiently recruited to mitochondria. Consistent with previous reports, the C431S mutant did not translocate to mitochondria, nor did the T240R mutant (Figure 1D). In PARK2 knockout cells, all mutants, including the C431S mutant, failed to form complexes (Figure 1E), suggesting that endogenous PARK2, even at low levels, can interfere with functional analysis of PARK2 mutants.
Figure 1. PARK2 mutants blocked in mitophagy have impaired complex formation. (A) Steady-state levels of PARK2 substrates in wild-type FLAG-PARK2 and the indicated variants. (B) Isolated mitochondria were solubilized and protein complexes were separated using BN-PAGE. Complexes containing PARK2 were detected by immunoblotting for PARK2. (C) Confirmation of PARK2 knockout. (D) PARK2 knockout cell lines were transiently transfected with wild-type FLAG-PARK2 and the corresponding mutant versions. Two days after transfection, cells were treated with 10 µM CCCP or DMSO for 3 hours. Cell lysates were separated into cytosol and mitochondria-enriched fractions and analyzed by SDS-PAGE and western blotting. (E) Mitochondria-enriched fractions from (D) were solubilized and separated by BN-PAGE or SDS-PAGE analysis followed by immunoblotting. (Callegari S, et al., 2017)
1. Parkinson's Disease Research: The PARK2 gene encodes parkin, a protein involved in the ubiquitin-proteasome system and mitochondrial quality control. Mutations in PARK2 are linked to juvenile Parkinson's disease. Using PARK2 Knockout HeLa cells, researchers can study the pathological mechanisms underlying Parkinson's disease, including mitochondrial dysfunction, oxidative stress, and proteasome impairment.
2. Drug Screening and Development: PARK2 Knockout HeLa cells can be employed for high-throughput screening of potential therapeutic compounds aiming to restore normal function or compensate for PARK2 loss. By evaluating the effects of various drugs on cell viability, mitochondrial function, and protein degradation, researchers can identify promising candidates for treating Parkinson's disease and other neurodegenerative disorders.
3. Mitochondrial Dysfunction Studies: Given the role of parkin in mitophagy (the autophagic degradation of damaged mitochondria), PARK2 Knockout HeLa cells serve as an essential model for studying processes related to mitochondrial quality control.
4. Autophagy and Proteostasis Research: PARK2 is implicated in the regulation of autophagy and proteostasis through the ubiquitin-proteasome system. PARK2 Knockout HeLa cells can thus provide a system for analyzing how disruptions in parkin function influence autophagic pathways and protein turnover, which is crucial for understanding various neurodegenerative and age-related diseases.
5. Cancer Research: Recent studies have suggested that PARK2 may function as a tumor suppressor. PARK2 Knockout HeLa cells can be utilized to explore the role of parkin in cancer biology, particularly in mechanisms of cell cycle regulation, apoptosis, and response to cellular stress.
6. Cellular and Molecular Biology Studies: PARK2 Knockout HeLa cells provide a versatile model for a wide array of cellular and molecular biology experiments. This includes studies on gene expression, signal transduction pathways, cellular metabolism, and interactions between different organelles.
For research use only. Not intended for any clinical use.
