Abstract:

It was to construct eukaryotic express vector PcDNA3.1/necdin and obtain the monoclonal P19 cell highly expressing necdin, and then detect the effect of overexpressed necdin on the cell proliferation of P19 cell. Total RNA was extracted from normal P19 cells. RT-PCR was used to amplify the aimed segments necdin which was then digested with EcoR Ⅰ and Xho Ⅰ and inserted into a eukaryotic expression plasmid PcDNA3.1 to construct PcDNA3.1/necdin. The constructed vector was transfected into P19 cells through lipofectamine2000-mediated transfer method. The transfected cells were treated with G418 until monoclonal cells appeared. Expression level of Necdin in monoclonal P19 cells was assayed by Western blot. CCK-8 method was utilized to detect the effect of overexpressed necdin on the cell proliferation of P19 cell.
Results showed that eukaryotic express vector PcDNA3.1/necdin was successfully constructed and obtained monoclonal P19 cells stably and highly-expressing necdin. Detection of cell proliferation found no obvious change in P19 cells highly expressing necdin. The study showed that overexpressed exogenous necdin have no obvious effect on the cell proliferation of P19 cells.

Key words: Necdin, Vector construction, Cell transfection, P19 embryonal carcinoma cells, Overexpression, Cell proliferation