[Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line].
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.
Gan
Zhi-Hua
ZH
Shanghai Institute of Hematology, Department of Hematology, Ruijin Hospital, Shanghai Jiaotong University Medical College, Shanghai 200025, China.
Chen
Yu
Y
Yan
Hua
H
Wang
Kan-Kan
KK
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D051880:Receptors, Notch / Q000378:metabolism*; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction; D014162:Transfection
D002135:Calcium-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C000606274:Jag1 protein, mouse; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; D051880:Receptors, Notch; D000072099:Serrate-Jagged Proteins