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Published on: July 22, 2016
[Construction of cyclin D1 recombinant plasmids and its expression in human nasopharyngeal carcinoma cells]
Zhi-quan Bai1, Hai-feng Zhang, Li-xin Chen
1Department of Physiology, Medical College of Jinan University, Guangzhou 510632, China.
Objective:
To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).
Methods:
The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.
Results:
Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting.
Conclusion:
We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.
