[Expression of eukaryotic expression plasmid pcDNA3/pacA and pcDNA3/pacP in mammalian cells]

Prior to the use of pcDNA3/pacA and pcDNA3/pacP in vivo, the transcription and expression products of these two eukaryotic expression plasmids in mammalian cells were detected.

The eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP were transfected into COS-7 cells respectively with liposome according to the manufacturer's protocol. In order to generate stable transfectants, after a 48 h incubation period in regular Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum, cells were treated with neomycin (1 mg.ml-1 of G418) and maintained under continuous selective pressure. G418-resistant colonies, which became visible after 2 weeks, were isolated and screened for expression of the vector-encoded protein. The control transfections were performed with the pcDNA3 vector and without recombinant as well as vector plasmid DNA. The mRNA transcriptions of the two insertional genes were detected by RT-PCR assay and their expression products were analysed by labelled-avidin-biotin enzymed-linked immunosorbent assay, flow cytometry and Western blotting. The cells transfected by pcDNA3 were used as the negative control.

1. The two eukaryotic expression plasmids could be correctly transcripted and translated under the control of the CMV immediate early promoter in mammalian cells. 2. The protein products could be detected in cell plasma, cell membrane and the culture supernatant.

The two eukaryotic expression plasmids pcDNA3/pacA and pcDNA3/pacP can express protein products which were encoded by insertional gene pac-A and pac-P in mammalian cells.