Transfected Stable Cell Lines
Reliable | High-Performance | Wide Rage
Precision reporter, kinase, immune receptor, biosimilar, Cas9, and knockout stable cell lines for diverse applications.
Cat. No. : CSC-RO0428
Host Cell : Ba/F3 Size : >1x106 frozen cells/vial
| Cat. No. | CSC-RO0428 |
| Description | This cell line is engineered to stably overexpress exogenous human TPR-MET fusion protein. |
| Target Gene | TPR-MET |
| Gene Species | Homo sapiens (Human) |
| Host Cell | Ba/F3 |
| Host Cell Species | Mus musculus (Mouse) |
| Applications | Drug screening and biological assays |
| Biosafety Level | 2 |
| Size | >1x10^6 frozen cells/vial, 1 mL |
| Stability | Stable in culture over a minimum of 10 passages |
| Quality Control | Negative for bacteria, yeast, fungi and mycoplasma. |
| Growth Conditions | 37 °C, 5% CO2 |
| Storage | Liquid nitrogen |
| Shipping | Dry ice |
| Thawing & Subculturing Instructions | 1. Thaw cells by gently swirling in a 37°C water bath. To limit contamination, do not submerge the O-ring and cap. 2. When cells are ~70% thawed (~1 min), transfer the vial into a biosafety cabinet, and wipe the surface with 70% ethanol. Allow tube to dry completely. 3. Transfer the cells gently into a 15 mL conical tube containing 10 mL of pre-warmed culture medium (without antibiotic selection marker). Centrifuge cells at ~125 x g for 5~7 min. 4. Remove supernatant without disturbing the pellet, and resuspend cells in 1 mL culture medium (without antibiotic selection marker). Transfer cells to a 6-well plate containing ~2 mL pre-warmed growth medium (without antibiotic selection marker) or a T25 flask containing 5 mL pre-warmed culture medium (without antibiotic selection marker). 5. Incubate the culture at 37°C with 5% CO2. 6. Subculture: split saturated culture 1:4 ~ 1:6 every 3 days; seed out at about 1~3 x 10^5 cells/mL. |
| Freeze Medium | Frozen with 70% medium, 20% FBS, 10% DMSO |
| Growth Properties | Suspension, round |
| Freezing Instructions | Cells are recommended to generate additional frozen stocks at early passages. Frozen stocks should be preserved in a designated cryopreservation medium or in 70% RPMI 1640 + 20% FBS + 10% DMSO (without antibiotic selection marker). 1. Prepare the freezing medium (70% RPMI 1640 + 20% FBS + 10% DMSO, without antibiotic selection marker) fresh immediately before use. 2. Keep the freezing medium on ice and label cryovials. 3. Transfer cells to a sterile, conical centrifuge tube, and count the cells. 4. Centrifuge the cells at 250 x g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3 x10^6 cells/ml in chilled freezing medium. 6. Aliquot 1 ml of the cell suspension into each cryovial. 7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer. 8. Transfer vials to liquid nitrogen for long-term storage. |
| 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 |
| Target Gene | TPR-MET |
The technique of Transcriptional Promoter Reporter (TPR) and its subsequent modifications, such as the Multicolor Expression Technology (MET), have revolutionized the field of cellular biology research. Originally conceived in the latter part of the 20th century, TPR marked a pivotal advancement in our ability to study gene expression dynamics within living cells. By integrating specific promoter regions with reporter genes, TPR facilitated the direct visualization and precise quantification of transcriptional activity, offering invaluable insights into the intricacies of gene regulation.
Building upon the foundation laid by TPR, the introduction of Multicolor Expression Technology (MET) represented a significant leap forward. MET's innovation lies in its capability to simultaneously monitor multiple gene promoters within a single cell, providing researchers with a comprehensive view of transcriptional events occurring within complex cellular environments. This breakthrough has greatly expanded our understanding of gene regulatory networks and their functional implications.
The development of the Human TPR-MET Stable Cell Line - BaF3 exemplifies the continual refinement of these methodologies. By integrating human-specific transcriptional regulatory elements with the MET technology, this stable cell line offers researchers a reliable and physiologically relevant model system for investigating gene expression dynamics in human cells. The availability of such a tool is instrumental in unraveling the complexities of human biology and has profound implications for various fields, including drug discovery, disease modeling, and regenerative medicine.
MET-targeted therapies are effective in MET-amplified and METex14 NSCLC, but face drug resistance challenges. Researchers assessed MET tyrosine kinase inhibitors (TKIs) in overcoming drug resistance in NSCLC. Using TPR-MET transformed Ba/F3 cell mutagenesis assays, they evaluated secondary MET mutations after TKI treatments. While individual TKIs showed distinct mutation profiles, combining type I/II TKIs (capmatinib and merestinib) resulted in no resistant clones in vitro. In vivo, this combination significantly reduced tumor growth compared to single TKIs. Simultaneous use of type I and type II MET TKIs may delay or diminish drug resistance mutations, offering a promising clinical approach.
Figure 1. Using the Human TPR-MET Stable Cell Line, distinct secondary MET mutations upon MET TKI treatment were studied. Mutagenized cells were treated until resistant clones emerged, facilitating clone counting. Sequencing revealed secondary MET TKD mutations, aiding in understanding drug resistance mechanisms. (Bahcall M, et al., 2022)
1. Signal Transduction Studies: Investigating downstream signaling pathways activated by TPR-MET fusion protein using BaF3 cells as a model system.
2. Targeted Therapy Evaluation: Assessing the potency and selectivity of novel therapeutic agents against TPR-MET fusion protein in BaF3 cells.
3. Mechanism Elucidation: Studying the molecular mechanisms underlying oncogenic transformation mediated by TPR-MET fusion through genetic manipulation in BaF3 cells.
4. Resistance Mechanism Exploration: Utilizing BaF3 cells expressing human TPR-MET to elucidate mechanisms of resistance to MET-targeted therapies in cancer treatment.
5. Preclinical Efficacy Testing: Conducting in vitro studies using BaF3 cells harboring TPR-MET to evaluate the preclinical efficacy of potential anti-MET therapeutics.
A: BaF3 cells were chosen for establishing the stable cell line expressing human TPR-MET fusion protein due to their lack of endogenous MET expression and their responsiveness to MET activation, providing a clean background for studying TPR-MET signaling. BaF3 cells also offer a suitable environment for investigating the oncogenic potential and drug sensitivity of TPR-MET.
A: The stability and expression level of human TPR-MET in the BaF3 stable cell line was confirmed and sustained through stable transfection techniques, followed by clonal selection to isolate cells with stable integration of TPR-MET. Expression of TPR-MET was validated using qPCR and western blot analysis. Continuous culture in the presence of appropriate selection agents ensured the maintenance of stable expression levels.
A: Functional characterization of human TPR-MET in the BaF3 stable cell line focused on its kinase activity and downstream signaling effects. This involved assessing the phosphorylation of key signaling molecules such as MAPK and AKT using western blotting. Additionally, cell proliferation and migration assays were conducted to evaluate the oncogenic potential and downstream effects of TPR-MET activation.
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Rock-solid stability. The Human TPR-MET Stable Cell Line in Ba/F3 cells keeps TPR-MET fusion protein expression consistent, ensuring reliable results in cancer research. With stable TPR-MET expression, I'm digging into oncogenic signaling pathways confidently, deepening our understanding of cancer biology.
Dependable to the core. This cell line exceeds expectations, giving me a sturdy platform for studying TPR-MET-targeted therapies and MET-driven cancers. It's supercharged my research, offering valuable insights into TPR-MET-mediated oncogenesis and potential cancer treatments.
Its stable expression makes experiments easier, speeding up data collection and analysis for breakthroughs in cancer pathogenesis.
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