Human CD46 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT2744
Host Cell: HEK293T Target Gene: CD46
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT2744 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human CD46 using CRISPR/Cas9. |
| Target Gene | CD46 |
| 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
CD46, also known as membrane cofactor protein (MCP), is a key protein encoded by the human CD46 gene. The gene responsible for CD46 is located on chromosome 1q32 and it produces a type I membrane protein. CD46 plays multiple roles in the complement system, acting as a regulatory protein. It protects host cells from damage by complement activation through its cofactor activity, facilitating the inactivation of complement components C3b and C4b by serum factor I. In addition to its role in the immune system, CD46 acts as a receptor for a variety of pathogens, including the Edmonston strain of measles virus, human herpesvirus type 6 (HHV-6), group B adenovirus, and pathogenic Neisseria. The protein structure of CD46 is adaptive and undergoes conformational changes upon interaction with these microbial factors, thereby facilitating their entry into cells.
Clinically, the importance of CD46 extends to a variety of pathological conditions. Due to its regulatory function, it is associated with diseases such as atypical hemolytic uremic syndrome. Expression of this protein has been linked to certain cancers, including medulloblastoma (a common childhood malignant brain tumor) and prostate cancer, making it a potential target for therapeutic interventions such as vaccines and antibody-drug conjugates. In addition, CD46 interacts with other proteins, including integrins such as CD9, CD29, and CD151, and helps regulate immune responses (including T cell-mediated immunity) and the production of cytokines such as interleukin 10. Its deficiency or dysfunction can lead to inflammatory diseases, suggesting a broader impact on immune regulation and homeostasis.
Lentiviral vectors (LVs) pseudotyped with measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to transduce hematopoietic stem and progenitor cells (HSPCs) more efficiently than LVs pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G). However, the use of H/F LVs is limited by the low vector titers produced. Studies here show that measles receptor (CD46) expression on H/F-transfected HEK293T vector-producing cells leads to fusion of adjacent cell membranes, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV. H/F LVs produced in CD46 knockout HEK293T cells produced vector titers that were 2-fold higher than LVs produced in CD46+ HEK293T cells. This resulted in an approximately 2- to 3-fold increase in the transduction efficiency of HSPCs, while significantly reducing target cell cytotoxicity caused by producer cell contaminants. Improved entry of H/F LV into HSPCs compared with VSV-G LV was also observed by confocal microscopy, with distinct entry mechanisms. Given that vector production is a major source of cost and variability in gene therapy clinical trials, the researchers propose that the use of CD46-null packaging cells may help address these challenges.
To determine whether CD46 null packaging cells could improve vector production, researchers generated H/F LV in CD46 WT and CD46 knockout HEK293T cells. Multinucleated syncytia formation was observed in WT cells 24 hours after transfection, and severe cytotoxicity developed after 3 days (Figure 1A). In contrast, CD46 knockout HEK293T showed no syncytia formation, limited cytotoxicity, and sustained cell proliferation over the same culture period (Figure 1B). Transfection of WT and CD46 knockout HEK293T cells with packaging plasmids for 2 days resulted in equivalent vector yields, as indicated by similar amounts of p24 protein.
Figure 1. H/F LV Production in CD46 Knockout HEK293T Cells Is Non-cytotoxic and Does Not Form Syncytia Observed in Wild-Type HEK293T Cells. (Ozog S, et al., 2019)
1. Viral infection research: CD46 is known to be a receptor for certain viruses. Knocking out CD46 in HEK293T cells allows researchers to study the effects of CD46 loss on viral entry and replication, helping to identify potential antiviral targets.
2. Immune response research: CD46 plays a role in regulating the complement system. Using CD46 knockout cells can help understand the immune evasion strategies of pathogens and develop therapies that modulate immune responses.
3. Cancer research: CD46 is often overexpressed in several types of cancer. Studying the role of CD46 in tumor progression and the tumor microenvironment can facilitate the development of targeted cancer therapies.
4. Biological pathway analysis: Understanding how CD46 loss affects various cellular pathways and processes can help to elucidate its role in normal physiology and disease states.
5. Therapeutic development: CD46 knockout models can be used to test new drugs and treatments designed to target CD46-related pathways or conditions, ultimately aiding in the discovery of new treatment options.
For research use only. Not intended for any clinical use.
