Cat.No. : CSC-RT0483
Host Cell: HEK293T Target Gene: CD47
Size: 2x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT0483 |
| Description | This cell line is a stable cell line with a homozygous knockout of human CD47 using CRISPR/Cas9. |
| Target Gene | CD47 |
| Gene ID | 961 |
| Genotype | CD47 (-/-) |
| Host Cell | HEK293T |
| Host Cell Species | Homo sapiens (Human) |
| Cell Type | Epithelial |
| Size | 2x10^6 cells/vial, 1mL |
| SequencingResult | Allele 1: 14 bp deletion Allele 2: 5 bp deletion |
| 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 |
CD47 is a ligand for SIRPα, an inhibitory receptor expressed by macrophages, dendritic cells, and natural killer (NK) cells, and therefore, transgenic overexpression of CD47 has been considered an effective approach to inhibit transplant rejection. However, the deleterious effects of CD47 signaling have been overlooked when exploring this approach. Here, researchers constructed mutant CD47 by replacing the transmembrane and intracellular domains with a membrane anchor (CD47-IgV). In both human and mouse cells, CD47-IgV was efficiently expressed on the cell surface and prevented phagocytosis in vitro and in vivo, but did not induce cell death or inhibit angiogenesis. In addition, hematopoietic stem cells expressing transgenic CD47-IgV showed no detectable alterations in engraftment or differentiation. This study provides a potentially effective method to achieve transgenic CD47 expression, which may facilitate the production of gene-edited pigs for xenotransplantation and low-immunogenic pluripotent stem cells for regenerative medicine.
In this study, flow cytometry analysis showed that, similar to hCD47-iso2, hCD47-IgV was efficiently expressed on the cell surface with no or minimal intracellular retention (Figure 1A). Furthermore, consistent with the critical role of the transmembrane and intracellular domains in transmitting CD47 signals, incubation with agonist anti-CD47 antibodies resulted in apoptosis of hCD47-iso2-expressing cells, but not hCD47-IgV-expressing cells (Figure 1B). Next, the researchers evaluated the protective effect of hCD47-IgV on phagocytosis. Researchers found that Jurkat cells expressing hCD47-IgV and hCD47-iso2 had significantly reduced levels of phagocytosis compared with CD47 Knockout (CD47KO) cells (Figure 1C). Flow cytometry (Figure 1D) and confocal analysis (Figure 1E) showed that cells expressing hCD47-IgV and hCD47-iso2 had significantly reduced levels of phagocytosis compared to CD47 Knockout (CD47KO) cells. In repeated experiments, the researchers sorted cells with different levels of hCD47 expression and found that the degree of protection seemed to correlate with the level of transgenic hCD47 expression in cells expressing hCD47-IgV and hCD47-iso2 (Figure 1F-1H). These results indicate that hCD47-IgV is as efficient as hCD47-iso2 in inhibiting phagocytosis in vitro.
Figure 1. Comparable protection against phagocytosis of Jurkat cells by transgenic expression of hCD47-IgV and CD47-iso2. (Xu, Lu, et al. 2024)
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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