Cloning and Expression of pCMV3-noggin-GFP Spark in Breast Cancer Cell Line, MCF-7

Introduction & 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. Methods: 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-noggin-GFP spark including GFP as a reporter gene, which was already transformed in DH5α bacterial cells and subjected to PCR by using the specific primers. Then, the transfected cells examined 24h post-electroporation by using epifluorescent microscopy. Results: pCMV3-noggin-GFP spark had been cloned successfully in DH5α and PCR confirmed the extracted plasmid contained the desired fragment (noggin +GFP sequences). Our results also showed that the highly expression of NOGGIN protein following transient transfection in MCF-7.Conclusion: pCMV3-noggin-GFP spark represents a promising cloning vector expressing NOGGIN protein in breast cancer cell line, MCF-7.