Cloning and Expression of Recombinant Human Noggin Protein Fused with GFP spark in Breast Cancer Cell line

Aghvami Tehrani, Azadeh (2019) Cloning and Expression of Recombinant Human Noggin Protein Fused with GFP spark in Breast Cancer Cell line. Masters thesis, University of Mohaghegh Ardabili.

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Abstract

Research Aim:Breast cancer metastasis is the expansion of cancer cells to tissues beyond where the tumor initially forms. During this process, the epithelial cells transform to mesenchymal cells (EMT) by different signaling pathway including TGFβ1 superfamily protein (such as BMPs). This study was aimed at cloning and overexpressing of NOGGIN protein, as a potent BMP antagonist in breast cancer cells. Research method:The human breast cancer cell line, MCF7 was grown in DMEMHG+10% FBS at 37oC with 5% CO2 to reach up to 70-80% confluency. The harvested cells underwent to transient transfection with pCMV3-NOG-GFPspark including GFP as a reporter gene gene which was already transformed in DH5α bacterial cells and subjected to PCR by using the specific primers.Then, the transfected cells examined24h post- electroporation by using epifluorescent microscopy. Findings&Conclusion:pCMV3-noggin-GFPspark had been cloned successfully in DH5α and PCR confirmed the extracted plasmid containing the desired fragment (noggin +GFP sequences). Our results also showed that the highly expression of NOGGIN protein following transient transfection in MCF-7 and expression of the gene were studied by the SDS-PAGE electrophoresis.

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