Suppression subtractive hybridization for screening of upregulated gene expressions in interferon-alpha transfected HepG2 cell line

AIM: To screen the upregulated genes in interferon-alpha(IFN-α) transfected HepG2 cells by suppression subtractive hybridization (SSH) technique with further investigation the molecular mechanism of IFN-α. METHODS: The mRNA was isolated from HepG2 cells transfected by pcDNA3.1 (-)-IFN-α and pcDNA.l (-) empty vector, and then cDNA was synthesized. After restriction enzyme Rsa I digestion, small sizes of cDNA were obtained. Then tester cDNA was subdivided into two portions and each was connected with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice,the PCR products were subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was earried out with E.Coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Rlast search after PCR. RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. Colony PCR showed that these clones contained 200-1 000 bp inserts. Thirty-five clones were analyzed by sequencing and bioinformatics. Seventeen known genes and one chromatic sequence were obtained. CONCLUSION: A subtractive cDNA library of genes upregulated in pcDNA3.1 (-)-IFN-αtransfected HepG2 cells by SSH technique was constructed successfully, which may bring some new important theoretical clues for studing the biological functions of IFN-α.