Human LRRK2 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT2781
Host Cell: HEK293T Target Gene: LRRK2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2781 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of LRRK2 using CRISPR/Cas9. |
| Target Gene | LRRK2 |
| Host Cell | HEK293T |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid Nitrogen |
| 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 LRRK2 gene, also known as leucine-rich repeat kinase 2, dardarin, and PARK8, is known for encoding a protein kinase involved in a variety of biological and enzymatic functions. Structurally, the LRRK2 protein is complex, with several distinct domains, including the armadillo repeat (ARM), ankyrin repeat (ANK), leucine-rich repeat (LRR) domain, kinase domain, RAS domain, GTPase domain, and WD40 domain. This structural diversity enables LRRK2 to interact with a variety of cellular components and affect a variety of cellular processes. The protein is primarily located in the cytoplasm but is also associated with the outer mitochondrial membrane, playing a crucial role in maintaining cellular function and integrity.
One of the most important aspects of LRRK2 is its association with Parkinson's disease (PD). Mutations in the LRRK2 gene, particularly the Gly2019Ser mutation, are associated with an increased risk of developing Parkinson's disease. This mutation enhances the kinase activity of the LRRK2 protein, leading to neurodegenerative changes that are characteristic of PD. Notably, the Gly2019Ser mutation is one of the most common genetic causes of Parkinson's disease and is particularly prevalent in certain ethnic groups, such as Ashkenazi Jews and North African Berbers. Additionally, the LRRK2 gene has been linked to other diseases, such as Crohn's disease. Understanding the function of LRRK2 and the impact of its mutations could help pave the way for the development of targeted therapies and treatments for diseases like Parkinson's disease.
Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the vulnerability of dopaminergic neurons to oxidative stress (OS), mitochondrial damage, and increased cell death in idiopathic and familial Parkinson's disease (PD). Here, LRRK2 knockout (KO) HEK-293 cells were used to assess the cellular response to the mitochondrial inhibitor complex I, rotenone (ROT), a well-known inducer of OS and cell death. The study found that exposure of HEK-293 LRRK2 WT cells to rotenone (ROT) resulted in a significant increase in intracellular reactive oxygen species (ROS); enhanced expression of tumor protein (TP53), p53 upregulated modulator of apoptosis (PUMA), and Parkin (PRKN); activation of caspase 3 (CASP3), DNA fragmentation and decreased mitochondrial membrane potential and PTEN induced putative kinase 1 (PINK1) when compared to untreated cells. Translocation of the cytoplasmic fission protein dynamin-related protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of outer membrane 20 (TOM20). Notably, HEK-293 LRRK2 KO cells treated with ROT showed unchanged OS and apoptotic markers. It was concluded that loss of LRRK2 renders HEK-293 resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. These observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.
To determine whether ROT induced apoptosis in both WT and KO cells, cells were exposed to ROT. ROT increased the activation of p-Ser65-cJUN (Figure 1A,C), TP53 (Figure 1D,F), PUMA (Figure 1G,I), and CASP3 (Figure 1J,L) by +171%, +2000%, +1950%, and +8000%, respectively, in HEK-293 LRRK2 WT cells compared to untreated WT cells. However, ROT did not induce appreciable expression of apoptotic markers in LRRK2 KO cells compared to untreated KO cells (Figure 1B,C,E,F,H,I,K,L). Similar observations were revealed by fluorescent microscopy (FM).
Figure 1. LRRK2 KO induces no activation of pro-apoptosis proteins under ROT stimuli. (Quintero-Espinosa D A, et al. 2023)
Applications of Human LRRK2 Knockout Cell Line-HEK293T
Neurodegenerative Disease Research: The Human LRRK2 Knockout Cell Line-HEK293T is extensively used in the study of neurodegenerative diseases, particularly Parkinson’s Disease. LRRK2 mutations are linked to both familial and sporadic forms of Parkinson’s disease. Knockout cell lines help researchers understand the underlying molecular mechanisms by studying the effects of LRRK2 deficiency.
Drug Screening and Development: This cell line serves as a crucial model for high-throughput screening and development of therapeutic compounds. By comparing the effects of various drugs on LRRK2 knockout versus wild-type HEK293T cells, researchers can identify potential treatments that target specific pathways influenced by LRRK2.
Cell Signaling Studies: LRRK2 is a kinase involved in numerous signaling pathways. Using the LRRK2 knockout HEK293T cell line allows scientists to dissect the role of LRRK2 in signaling cascades, understanding its impact on cellular processes such as autophagy, inflammation, and immune response.
Protein Interaction Analysis: Researchers use this knockout cell line to investigate protein-protein interactions involving LRRK2. This is pivotal in identifying interaction partners and understanding the functional consequences of these interactions in the context of cellular physiology and pathology.
For research use only. Not intended for any clinical use.
