Human CD47 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT0483
Host Cell: HEK293T Target Gene: CD47
Size: 2x10^6 cells/vial, 1mL Validation: Sequencing
Inquire for Price
Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT0483 |
| Cell Line Information | 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 |
| Cell Type | Epithelial |
| Size | 2x10^6 cells/vial, 1mL |
| Sequencing Result | 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 |
Inquiry
Background
Case Study
Applications
CD47, also known as integrin-associated protein (IAP), is a transmembrane protein that plays a key role in a variety of physiological and pathological processes. It is widely expressed in human tissues, including immune cells, and its important function is to send a "don't eat me" signal to macrophages and other phagocytes by interacting with signal regulatory protein α (SIRPα). This interaction prevents cellular phagocytosis and is an important checkpoint in the immune system.
In recent years, CD47 has attracted great attention in oncology because it is overexpressed on the surface of many cancer cells. Tumors use the CD47-SIRPα interaction to avoid being phagocytosed by macrophages, effectively evading the natural surveillance mechanism of the immune system. Therapeutic strategies targeting CD47 have been developed to block its interaction with SIRPα, thereby enhancing the phagocytosis and subsequent destruction of cancer cells by the immune system. Monoclonal antibodies against CD47, such as Hu5F9-G4, are among the promising drug candidates currently being studied clinically. These approaches aim to disrupt the protective barrier surrounding tumors, making them more vulnerable to immune attack while ideally protecting normal cells.
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)
The knockout of CD47 in 293T cells provides an innovative tool for scientific research with broad applications.
Cancer Research: CD47 is often termed the "don't eat me" signal because it helps cancer cells evade immune detection. By using CD47 knockout 293T cells, researchers can study the mechanisms behind immune evasion and develop new anticancer therapies targeting the CD47-SIRPα signaling pathway.
Immunotherapy Development: The CD47 knockout model helps in the development of novel immunotherapies that aim to enhance macrophage-mediated phagocytosis of tumor cells. This is particularly useful for preclinical testing of CD47-blocking antibodies and other agents.
Drug Screening and Testing: CD47 knockout 293T cells are utilized to screen for small molecules or biological agents that can restore or inhibit the function of CD47, providing insights for potential therapeutic candidates.
Cell Migration and Adhesion Studies: CD47 is involved in cell migration and adhesion processes. Knockout cell lines allow scientists to dissect the role of CD47 in these cellular processes, potentially uncovering new aspects of tissue regeneration and wound healing.
Signal Transduction Research: CD47 interacts with various integrins and other transmembrane proteins, influencing a multitude of signaling pathways. Using CD47 knockout 293T cells helps to map these pathways more precisely, contributing to the broader understanding of cell signaling dynamics.
For research use only. Not intended for any clinical use.
