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Updated: Jun 10, 2026
Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression
Published on: March 5, 2013
[Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line]
Zhi-Hua Gan1, Yu Chen, Hua Yan
1Shanghai Institute of Hematology, Department of Hematology, Ruijin Hospital, Shanghai Jiaotong University Medical College, Shanghai 200025, China.
Abstract:
Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.
Insights
Researchers developed a Jagged-1 over-expressing NIH-3T3 cell line. This model aids in studying Notch signaling in drug resistance and identifying new therapeutic targets.
Area of Science:
- Cell Biology
- Molecular Biology
- Signaling Pathways
Background:
- The Notch signaling pathway regulates hematopoietic cell proliferation and differentiation.
- Jagged-1 is a key ligand in Notch signaling, crucial for cell-cell interactions.
- Understanding Notch signaling in bone marrow stromal cells is vital for drug resistance mechanisms.
Purpose of the Study:
- To investigate the biological effects of Jagged-1 and Notch signaling.
- To elucidate the role of Notch signaling in bone marrow stromal cell-mediated drug resistance.
- To establish a cellular model for studying these mechanisms.
Main Methods:
- Cloning the Jagged-1 gene into a eukaryotic expression vector (pEGFP-IRES2-Jagged-1).
- Transfecting the vector into NIH-3T3 cells.
- Confirming Jagged-1 overexpression via Western blot and flow cytometry.
- Establishing a monoclonal cell line using selective medium and limiting dilution.
Main Results:
- Successful construction of the pEGFP-IRES2-Jagged-1 eukaryotic expression vector.
- Creation of a stable, monoclonal NIH-3T3 cell line overexpressing Jagged-1 protein.
- Validation of Jagged-1 overexpression in the established cell line.
Conclusions:
- The developed cell line provides a robust model for Notch signaling research.
- This model facilitates further investigation into drug resistance mechanisms.
- It offers potential for discovering novel drug targets in cancer therapy.
