Human PARP1 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT1113
Host Cell: HEK293T Target Gene: PARP1
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1113 |
| Cell Line Information | A stable cell line with a homozygous knockout of human PARP1 using CRISPR/Cas9. |
| Target Gene | PARP1 |
| Host Cell | HEK293T |
| Shipping | 10^6 cells/tube |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene ID | 142 |
| 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
PARP1, or poly (ADP-ribose) polymerase 1, is a key enzyme in the PARP family of proteins that plays an important role in various cellular processes, especially in maintaining genomic integrity. The enzyme is best known for its involvement in the DNA repair process, especially in the case of single-strand breaks (SSBs). When DNA damage occurs, PARP1 quickly detects the DNA strand break and binds to the DNA at the site of damage. Once attached, PARP1 catalyzes the addition of ADP-ribose units from NAD+ to target proteins, including itself, in a process called poly ADP-ribosylation. This post-translational modification signals the site and recruits other DNA repair machinery, promoting efficient and rapid repair of damaged DNA. Through this mechanism, PARP1 helps maintain genomic stability and prevents mutations that can lead to diseases such as cancer.
In addition to DNA repair, PARP1 is involved in a variety of other cellular mechanisms. It regulates transcription by modifying chromatin structure, which affects gene expression. It is also involved in regulating cell death pathways, inflammation, and even cellular stress responses. Inhibitors of PARP1, or PARP inhibitors (PARPi), have emerged as promising therapeutic agents, particularly for cancers with defective homologous recombination (HR) repair mechanisms, such as BRCA1 or BRCA2 mutant cancers. These tumors rely heavily on PARP-mediated DNA repair, and inhibition of PARP1 can lead to synthetic lethality, selectively killing cancer cells while sparing normal cells.
In human melanoma, PARP-1 and HIF-1α expression are tightly linked. Upon hypoxic stimulation, poly(ADP-ribose) (PAR) is synthesized and HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation of specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach, researchers demonstrate that PARP-1 dictates hypoxia-dependent HIF recruitment to chromatin at a range of HIF-regulated genes, while analysis of HIF-binding motifs (RCGTG) reveals that recognition of hypoxia-responsive elements is restricted in the absence of PARP-1. As a result, cells are less adaptable to hypoxia and exhibit reduced fitness during hypoxia induction. These data describe a fine-tuned regulation of HIF activation by PARP-1/PARylation and suggest that PARP inhibitors may have therapeutic potential for cancer types that exhibit HIF-1α hyperactivation.
ChIP-Seq analysis was performed on HEK 293T WT and HEK 293T PARP-1 knockout (PARP-1 KO) cells during early hypoxia (4 hours). ChIP-Seq showed that the majority of HIF-1α binding peaks detected were located within the first kilobase upstream of the translation start site: 48.16% for wild type and 43.9% for PARP-1 KO (Figure 1), which includes the 5′-UTR and promoter region of the gene. Statistical analysis was completed to eliminate non-significant peaks and compare them to input and normoxic control signals. 123 peaks were detected in hypoxic wild type cells and 68 peaks were detected in PARP-1 KO cells.
Figure 1. Chip-seq analysis of HIF-1α capacity to bind the promoters of its target genes on WT vs. PARP-1 knockout cells. (Martí, Juan Manuel, et al. 2021)
PARP1 is a key enzyme involved in the DNA repair process, and knocking out this enzyme provides insights into a variety of cellular mechanisms and disease models. Here are some important applications:
Cancer Research: PARP1 knockout in HEK293T cells provides an important tool for studying the role of PARP1 in cancer development and treatment, especially in understanding DNA repair mechanisms and the efficacy of PARP inhibitors in cancer treatment.
Drug Screening and Development: This cell line is very useful for high-throughput screening of potential PARP inhibitors, helping to identify novel therapeutic agents and assess their effectiveness without interfering with endogenous PARP1 activity.
Genomic Stability Studies: Researchers use these knockout cells to study pathways involving PARP1 in maintaining genomic integrity, studying how the loss of PARP1 affects DNA damage response and repair mechanisms.
Pathway Analysis: The loss of PARP1 provides an ideal model for dissecting the cellular pathways affected by PARP1, including those involved in apoptosis, necrosis, and cell signaling, allowing for a clearer understanding of its role in various biological processes.
Neurological Research: Given the role of PARP1 in neuronal cell death and neuroinflammation, this knockout cell line can be used as a model to explore neurodegenerative diseases.
For research use only. Not intended for any clinical use.
