Human TMEM165 Knockout Cell Line-HEK293T
Cat.No. : CSC-RT1103
Host Cell: HEK293T Target Gene: TMEM165
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
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Cell Line Information
Cell Culture Information
Safety and Packaging
| Cat. No. | CSC-RT1103 |
| Cell Line Information | A stable cell line with a homozygous knockout of human TMEM165 using CRISPR/Cas9. |
| Target Gene | TMEM165 |
| Host Cell | HEK293T |
| Shipping | 10^6 cells/tube |
| Storage | Liquid nitrogen |
| Species | Human |
| Gene ID | 55858 |
| 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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Case Study
TMEM165 is associated with a rare human genetic disease named TMEM165-CDG (congenital disorder of glycosylation). It is localized to the Golgi apparatus, is highly conserved during evolution, and belongs to uncharacterized protein family 0016 (UPF0016). The use of homologous TMEM165 knockout HEK293 cells was crucial to reveal the role of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor for many glycosylation enzymes. Severe Golgi glycosylation defects were observed in TMEM165 knockout human embryonic kidney (KO HEK) 293 cells that could be rescued by exogenous manganese supplementation. Interestingly, here the researchers demonstrate that the observed Golgi glycosylation defects are critically dependent on fetal bovine serum, especially its manganese levels. These results also suggest that iron and/or galactose can modulate the glycosylation defects observed in TMEM165 knockout HEK293 cells.
Previous studies have shown that supplementation with galactose can suppress some of the glycosylation defects of TMEM165-CDG. Here, the researchers wondered whether this suppression also depended on the source of the FBS. To tackle this point, TMEM165 KO HEK293 cells were cultured in two different FBS (FBS 2 or FBS 4) and supplemented with 1 μM MnCl2, 1 mM galactose or 1 μM MnCl2 + 1 mM galactose. Intriguingly, all treatments resulted in fully glycosylated forms of LAMP2 in TMEM165 KO HEK293 cells cultured in FBS 2 (Figure 1A). The result was completely different in cells grown in FBS 4 (Figure 1B). In the latter condition, galactose or Mn2+ supplementation poorly rescued LAMP2 glycosylation (Figure 1B). However, the combination of these two factors rescued the fully glycosylated form of LAMP2 (Figure 1B). This result suggests that, depending on the source of FBS, Mn2+ can enhance the effect of galactose on suppressing LAMP2 glycosylation defects (and vice versa). These results emphasize the critical importance of serum content on glycomic results.
Figure 1. The suppression of LAMP2 glycosylation defect by galactose supplementation depends on the FBS used for cell culture. (Vicogne D, et al., 2020)
For research use only. Not intended for any clinical use.
