Cat.No. : CSC-RT2707
Host Cell: CHO-K1 Target Gene: Fut8
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2707 |
| Description | This cell is a stable cell line with a homozygous knockout of Fut8 using CRISPR/Cas9. |
| Target Gene | Fut8 |
| Host Cell | CHO-K1 |
| Host Cell Species | Cricetulus griseus (Chinese hamster) |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid nirtogen |
| 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. |
| 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 |
α1,6-fucosyltransferase (encoded by the FUT8 gene) is a key enzyme in mammalian cells that transfers fucose to the innermost GlcNAc residue of the N-glycan via α-1,6 linkages. The presence of core fucose on the Fc region of antibodies can inhibit antibody-dependent cellular cytotoxicity (ADCC) and reduce the therapeutic efficiency of antibodies in vivo. Chinese hamster ovary (CHO) cells are a major production platform for biopharmaceutical manufacturing. Therefore, it is advantageous to generate a FUT8 knockout (FUT8KO) CHO cell line that can be used to produce fully non-fucosylated antibodies.
To understand the role of FUT8 in CHO cell glycosylation, researchers performed a large-scale glycoproteomic study using the FUT8 knockout CHO cell line. A total of 7,127 unique intact glycopeptides (IGPs) containing N-linked glycosyl sites, 928 glycosyl sites, and 442 glycoproteins were identified from FUT8 knockout (FUT8KO) and WT CHO cells. In addition, 28.62% of 442 identified glycoproteins and 26.69% of 928 identified glycosylation sites were significantly changed in FUT8KO CHO compared with wild-type CHO cells. In addition, a decrease in fucosylation content was observed in FUT8KO cells, with the core fucosylated glycans almost disappearing, which was the effect of FUT8 knockout. Meanwhile, a total of 51 glycosylation-related enzymes were quantified in both cell types, 16 of which were significantly altered in FUT8KO cells. These glycoproteomic results revealed that the knockout of FUT8 not only affected the core fucosylation of proteins, but also altered other glycosylation synthesis processes and changed the relative abundance of protein glycosylation.
Figure 1. Changes in fucosylated glycoproteins in FUT8KO CHO cells. (A) Heat map of fucosylated and core-fucosylated glycoproteins in WT and FUT8KO CHO cells. (B) The percentage of core-fucosylated glycoproteins among fucosylated glycoproteins in WT and FUT8KO CHO cells. (C) Changes in fucosylation of protein CD166 (7 glycosites) in CHO cells with FUT8KO. (Yang G, et al., 2021)
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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We have used the Fut8 Knockout Cell Line CHO-K1 in various assays, and the cells have consistently performed beyond our expectations.
The Fut8 Knockout CHO-K1 cells have proven to be incredibly useful for our glycoengineering projects. The quality and consistency of the cells are outstanding, and the knockout phenotype is stable even after multiple passages.
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