Abstract:

BACKGROUND: Studies demonstrated that Snai1 has been shown to negative interact with E-cadherin in tumor tissue and combine with CDH1 promoter region box motif, thus, suppress the CDH1 genetic transcription. 
OBJECTIVE: To construct snai1 eukaryotic expression vector and to establish a stable transfective cell line.
METHODS: Complete exon sequence based on the sequence of snai1 in the GenBank was designed and inserted into the plasmid Pmd-18T, recombining the plasmid Pmd-18T/snai1, which was transformed into E.coli DH5 and selected with ampicillin. The positive clones containing the recombinant Plasmid Pmd-18T/snai1 were sequenced and digested by restriction endonucleases EcoRI and XhoI.The digestion product of EcoRI and XhoI was purified and inserted into the eukaryotic expression vector pcDNA3.1(+) to construct pcDNA3.1(+)/Snai1,which was identified by sequencing and transfected into adenocarcinoma of colon HT-29 cells. The HT-29 cells with stable expression of snai1 were selected by G418 and confirmed by RT-PCR and Western blot.
RESULTS AND CONCLUSION: The eukaryotic expression vector pcDNA3.1(+)/snai1 was constructed successfully. HT-29 cells stably expressing snai1 were selected by G418 with 600 mg/L at 21 days. The successful construction of the plasmid of pcDNA3.1(+)/snai1 could significantly up-regulate the expression of snai1in colon neoplasms cells HT-29.