Cat.No. : CSC-RT2744
Host Cell: HEK293T Target Gene: CD46
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2744 |
| Description | This cell is a stable cell line with a homozygous knockout of human CD46 using CRISPR/Cas9. |
| Target Gene | CD46 |
| 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 |
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)
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.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
Great buy! The CD46 knockout cells are valuable tools in studying the immune system and investigating various diseases.
By analyzing CD46 knockout cells, we can unravel the precise signaling events that occur in the absence of CD46. Good experimental results were obtained.
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