Human CD81 Knockout Cell Line-HEK293
Cat.No. : CSC-RT2790
Host Cell: HEK293 Target Gene: CD81
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
Inquire for Price
Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2790 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human CD81 using CRISPR/Cas9. |
| Target Gene | CD81 |
| Host Cell | HEK293 |
| 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 |
Inquiry
Background
Case Study
Applications
CD81, also known as TAPA-1 (target of antiproliferative antibody 1) or tetraspanin-28, is a member of the transmembrane 4 superfamily, commonly referred to as the tetraspanin family. The CD81 gene is located on the short arm of chromosome 11 (11p15.5). CD81 is a cell surface glycoprotein that normally forms a complex with integrins and other proteins. It has been implicated in various cellular processes, including muscle cell fusion, maintenance of myotubes, and potentially signal transduction mechanisms. In addition, CD81 forms complexes with other molecules on B cells, such as CD19, CD21, and Leu-13, and helps regulate immune responses by binding to proteins such as CD4 and CD8 on T cells.
Clinically, CD81 plays a key role in the entry of hepatitis C virus (HCV) into cells by binding to the high-affinity HCV E2 glycoprotein dimer. This interaction is fundamental to the mechanism of HCV infection, as CD81-E2 binding facilitates viral entry. In addition to HCV, CD81 has been implicated in liver invasion by Plasmodium. Furthermore, CD81 is involved in HIV replication, serving as a platform for virion assembly and release. Interestingly, while antibodies against CD81 can downregulate HIV production, viruses lacking CD81 are more infectious, highlighting its complex role in viral pathogenesis.
The HCV pseudoparticle (HCVpp) system is a widely used platform to study cellular entry, screen for novel entry inhibitors, evaluate the phenotypes of E1 and E2 glycoproteins observed clinically, and most relevantly characterize the breadth of neutralizing antibodies induced in patients following vaccination and natural infection. Despite this, some patient-derived clones produce pseudoparticles that are either non-infectious or too low in infectivity for meaningful phenotypic analysis. Here, we show that endogenous expression of CD81, an HCV receptor and cognate binding partner for E2, in producer HEK 293T cells is deleterious to the infectivity of HCVpp recovered from most strains. Clones generated in CD81 knockout HEK293T cells are antigenically very similar to those generated from their matched parental cells and appear to follow the recognized HCV entry pathway. Deletion of CD81 does not significantly increase recovered titers of soluble E2 (sE2). The researchers also found that sE2 produced by Freestyle 293-F (293-F) cells contained mostly complex glycans, whereas sE2 produced by 293T displayed a heterogeneous mixture of complex glycans and high-mannose or mixed glycans. Furthermore, sE2 produced in 293T cells was superior in antigenicity; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that the antigenicity of sE2 produced in 293T and 293-F cells is not identical.
Here, researchers observed that HCVpp harvested from both CD81 knockout 293T lines had higher infection rates across the panel compared to matched viruses prepared in parental cells (Figure 2b). Improved infection was significantly more pronounced with HCVpp produced in CD81 knockout HEK293T cells compared to equivalents prepared in parental cells (Figure 1c). Finally, researchers prepared a panel of HCVpp expressing E1E2 in CD81 knockout HEK293T (293TCD81KO) cells observed in HCV+ individuals in the Australian Prison Hepatitis C Incidence and Transmission Study (HITS-p) cohort. Upon infection readout, an increase in the signal-to-noise ratio (S/N) was observed for the majority of screened clones prepared in 293TCD81KO cells. Surprisingly, 5 of the 22 screened clones that were previously identified as non-infectious were now infectious (S/N ≥5), and 8 of the additional 22 screened clones had at least a 5-fold increase in S/N. Finally, a comparison of the calculated S/N of all screened clones in all three panels showed an increasing trend in infectivity after HCVpp production in 293TCD81KO cells (Figure 1d). Taken together, the 293TCD81KO cells produced here can be a useful resource for HCV research.
Figure 1. HCVpp made in CD81 knock-out 293T cells exhibit enhanced infectivity. (Kalemera M D, et al., 2021)
Applications of Human CD81 Knockout Cell Line-HEK293
1. Viral Entry Studies: The human CD81 protein is known to play a crucial role in the entry of several viruses, including the Hepatitis C virus (HCV). Using CD81 knockout HEK293 cells, researchers can investigate the specific mechanisms by which viruses exploit CD81 for entry into host cells.
2. Cancer Research: CD81 has been implicated in various cancer-related processes, including cell adhesion, migration, and proliferation. HEK293 cells deficient in CD81 are valuable tools for studying how the absence of this protein affects tumor growth and metastasis. By analyzing these knockout cells, scientists can identify potential therapeutic targets and strategies for cancer treatment.
3. Drug Screening and Development: The CD81 knockout HEK293 cell line can be employed in high-throughput drug screening programs to identify compounds that interact specifically with CD81-dependent pathways.
4. Exosome Research: CD81 is a marker protein for exosomes, which are small vesicles secreted by cells that play a role in cell-to-cell communication. CD81 knockout HEK293 cells can be used to study the biogenesis, release, and function of exosomes.
5. Protein-Protein Interaction Studies: The absence of CD81 provides an opportunity to study how this protein interacts with other proteins at the cellular membrane. Utilizing CD81 knockout cells, researchers can perform co-immunoprecipitation and other biochemical assays to identify and characterize novel protein partners, shedding light on the complex network of cell-surface interactions.
For research use only. Not intended for any clinical use.
