Construction of recombinant MT1F overexpressed plasmids and validation of their transfection to RKO colon cancer cells

Objective To construct the recombinant MT1 F overexpressed plasmids and transfect them in RKO colon cancer cells.Methods The expression of MT1 F mRNA in six colon cancer cell lines was detected by using real-time polymerase chain reaction (PCR).MT1 F coding regions were synthesized to splice whole gene and then cloned into Cl terminus of pEGFP or pcDNA3.1( + ) vector to construct recombinant plasmids.The plasmids with 500 μl Lipofectamine were transfected into RKO cells after DNA sequencing,then screened by G418 (400 mg/L).The efficiencies of MT1F expression were validated by Western blotting ( mouse anti-human MT mAb 1 ∶ 500,mouse anti-GFP mAb 1 ∶ 1000).Results The lowest expression of MT1F mRNA was seen in RKO cells.The sequencing results validated the well recombinant plasmids.MT1 F mRNA in transient and stable transfeted RKO cells was upregulated about to 600 and 2000 fold,respectivly,and the 15 kD metallothionein proteins were detected.Conclusion The recombinant MT1F overexpressed plasmids were successfully constructed,and the stable RKO cells with ectopic MT1 F transfection were obtained. Key words: MT1F;  Colon carcinoma;  Gene transfection;  Plasmid