Cat.No. : CSC-RT2764
Host Cell: Hela Target Gene: TMEM165
Size: 1x10^6 cells/vial, 1mL Validation: Sequencing
| Cat. No. | CSC-RT2764 |
| Description | This cell is a stable cell line with a homozygous knockout of human TMEM165 using CRISPR/Cas9. |
| Target Gene | TMEM165 |
| Host Cell | Hela |
| Host Cell Species | Homo sapiens (Human) |
| Size Form | 1 vial (>10^6 cell/vial) |
| Shipping | Dry ice package |
| Storage | Liquid Nitrogen |
| 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 |
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)
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.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
We have been using the Human TMEM165 Knockout Cell Line-Hela for over six months in our laboratory, and the consistency and reliability of the results are outstanding.
This knockout cell line has significantly streamlined our experimental workflow, reducing variability and enhancing our data quality. Highly recommend it to anyone studying glycoprotein biosynthesis.
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