Construction of E2F-1 gene eukaryotic expression vector and selection of Genectin-resistant gastric carcinoma cells

Objective To construct an eukaryotic expression vector containing E2F-1 gene and transfect the vector into gastric cancer cell line MGC-803.Methods Total RNA was extracted from normal human liver.E2F-1 cDNA was cloned by using RT-PCR method and transferred into eukaryotic expression vector pCMV-HA2 after digested by restrictive endonuclease enzyme Hind Ⅲ and EcoRⅠ.Then pCMV-E2F-1-HA2 was transfected into MGC-803 cells with Lipofectamine 2000.The genectin-resistant cells were selected and harvested in medium containing G418.RT-PCR and Western blot were used to identify the expression of E2F-1.Results pCMV-E2F-1-HA2 prospective fragments was obtained and the result of sequencing was completely consistent with the reported E2F-1 gene sequences.Cell clones with resistance to Genetecin were obtained and amplified.Experimental results of RT-PCR and Western blot confirmed that the recombinant vector pCMV-E2F-1-HA2 was successfully transfected into gastric cancer cell line MGC-803 and stably overexpressed the E2F-1 gene.Conclusion Eukaryotic expression vector pCMV-E2F-1-HA2 had been successfully constructed.Gastric cancer cell line MGC-803/E2F-1 with stable overexpression of the E2F-1 gene was established.