Human TMEM165 Knockout Cell Line-Hela
Cat.No. : CSC-RT2764
Host Cell: Hela 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-RT2764 |
| Cell Line Information | This cell is a stable cell line with a homozygous knockout of human TMEM165 using CRISPR/Cas9. |
| Target Gene | TMEM165 |
| Host Cell | Hela |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid Nitrogen |
| Species | Human |
| 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 1:4-1:6. |
| 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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Background
Case Study
Applications
TMEM165, also known as transmembrane protein 165, is a protein encoded by the human TMEM165 gene. The TMEM165 gene is located on chromosome 4. TMEM165 is highly expressed in a variety of tissues, including the corpus callosum, gastric mucosa, vagus nerve, and lung tissue. The TMEM165 protein is involved in maintaining cellular homeostasis and is essential in the early secretory pathway, particularly in Golgi function and glycosylation. Glycosylation is the process by which sugars are chemically attached to proteins to form glycoproteins, which is essential for proper protein folding, stability, and cell signaling.
Mutations in the TMEM165 gene are associated with a rare genetic disorder, congenital disorder of glycosylation type II-K (CDG-IIK). This disease is characterized by a wide range of symptoms, including developmental delay, growth retardation, and multisystem involvement, such as liver dysfunction, skeletal abnormalities, and immune deficiency. The disease is caused by a defect in the glycosylation pathway, resulting in improper protein folding and dysfunction.
The TMEM165 gene encodes a multipass membrane protein located in the Golgi apparatus that is associated with innate disorders of glycosylation. Previous studies have shown that TMEM165 is a potential biomarker for breast cancer. TMEM165 protein was not detected in non-malignant matched breast tissues, but was detected by mass spectrometry in invasive ductal breast cancer tissues. Here, researchers investigated the expression of TMEM165 in early invasive ductal carcinoma and ductal carcinoma in situ cases. CRISPR/Cas9 knockout of TMEM165 was created in the human invasive breast cancer cell line MDAMB231. These results showed that removal of TMEM165 in these cells resulted in a significant reduction in cell migration, tumor growth, and tumor vascularization in vivo. In addition, the researchers found that TMEM165 expression altered the glycosylation of breast cancer cells, and these changes promoted breast cancer invasion and growth by altering the expression levels of key glycoproteins involved in regulating epithelial-mesenchymal transition, such as E-cadherin.
Here, the researchers evaluated the NCSTN glycoforms in MDAMB231, HEK293T, and HeLa TMEM165 knockout cell lines and their control cells. HEK293T TMEM165 knockout cells and HeLa TMEM165 knockout cells displayed NCSTN glycoforms that migrated to a lower molecular weight than MDAMB231 breast cancer TMEM165 knockout cells (Figure 1B). These results suggest that the NCSTN glycoprotein in MDAMB231 breast cancer cells has different glycosylation compared to HEK293T and HeLa cells. To rescue the altered NCSTN glycoforms in TMEM165 knockout cells, TMEM165WT plasmid and empty vector were transiently transfected into MDAMB231 cells. These results further confirmed that the altered glycosylation pattern observed in NCSTN was restored only in TMEM165 knockout cells after transfection with TMEM165WT plasmid but not vector (Figure 1C). Interestingly, when CDG mutant forms of TMEM165 (R126H and G304R) were transiently expressed in MDAMB231 cells, N-linked glycosylation was also present in the NCSTN glycoform, as observed in control cells ( Figure 1D ). These results suggest that the functionally disruptive changes in TMEM165 that occur in CDG patients do not disrupt the function of TMEM165 in MDAMB231 breast cancer cells, as these mutants are able to induce the nicastrin glycoform observed in MDAMB231 control cells.
Figure 1. TMEM165 expression levels alters N-linked glycosylation. (Murali P, et al., 2020)
1. Functional studies of TMEM165: TMEM165 knockout Hela cell lines allow researchers to study the physiological and molecular functions of the TMEM165 gene. By comparing knockout cells to normal cells, scientists can study how the loss of TMEM165 affects cellular processes and determine its role in various biological pathways.
2. Glycosylation studies: TMEM165 is known to be involved in glycosylation, a critical cellular process in which sugars are attached to proteins and lipids. Using the TMEM165 KO cell line, researchers can study the effects of TMEM165 loss on glycosylation patterns, helping to understand diseases associated with glycosylation defects, such as congenital disorders of glycosylation (CDG).
3. Disease modeling: Knockout cell lines can be used to model human diseases, especially those associated with TMEM165 mutations. By studying how these cells respond under different conditions, researchers can gain insight into TMEM165-related pathologies, laying the foundation for therapeutic development.
4. Drug screening and development: The TMEM165 KO Hela cell line is a valuable model for screening potential drug candidates. Compounds that rescue or recapitulate the effects of TMEM165 deficiency can be identified and developed as treatments for diseases associated with mutations in this gene.
5. Cell pathway analysis: TMEM165 knockout Hela cells are used to dissect cellular signaling pathways. By studying changes in signaling cascades following TMEM165 loss, researchers can map out pathways in which TMEM165 participates, leading to a better understanding of cellular function and regulation.
For research use only. Not intended for any clinical use.
