Cloneing and screening transregulated genes of XTP6 by SSH

Objective:To screen and identify human genes transactivated by XTP6 by constructing a cDNA subtractive library with suppression subtractive hybridization(SSH) technique.Methods:Suppression subtractive hybridization and bioinformatics techniques were used for screening and cloning of the target genes transactivated by XTP6 protein.The mRNA was isolated from HepG2 cells transfected pcDNA3.1(-)-XTP6 and pcDNA3.1(-) empty vector,and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups.After restriction enzyme Rsa I digestion,small sizes cDNAs were obtained.Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2,respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR,the second PCR production was subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library.Amplification of the library was transformed into E.coli.The cDNA was sequenced and analyzed in GenBank with Blastn search after PCR.Results: The subtractive library of genes transactivated by XTP6 was constructed successfully.The amplified library contains 70 positive clones containing 200-1000 bp inserts.Sequence analysis was performed in 20 clones randomly,and the full length sequences were obtained with bioinformatics method.Altogether 11 coding sequences were gotten.Conclusion:The obtained sequences may be target genes transactivated by XTP6,and some genes coding proteins involved in cell cycle regulation,biological metabolism and inflammation repair.