Cat.No. : CSC-RT2781
Host Cell: HEK293T Target Gene: LRRK2
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2781 |
| Description | This cell is a stable cell line with a homozygous knockout of LRRK2 using CRISPR/Cas9. |
| Target Gene | LRRK2 |
| Host Cell | HEK293T |
| 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 1:3-1:6. |
| 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 |
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)
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.
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As a neurodegenerative disease researcher, having reliable cell lines is crucial for my work. The Human LRRK2 Knockout Cell Line-HEK293T has consistently produced reproducible results, significantly enhancing the validity of my experimental data.
The LRRK2 Knockout HEK293T cells have been instrumental in our drug screening assays. The precise knockout of the LRRK2 gene allows us to better understand its role and screen for potential therapeutic compounds.
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