ABSTRACT
Objective To develop a rapid method for construction of RNAi vector.Methods After inversely inserting the U6 and H1 polymerase Ⅲ promoters into the pBluescriptⅡ backbone vector,only with two short reverse complementary oligo nucleotides,the RNAi vector with one interference cassette could be constructed.Using two isoaudamers MunⅠ and EcoRⅠ with sticky ends,several cassettes could be fused together to form the ultimate multiple-site targeting RNAi vector.Using this method,we constructed RNAi vectors targeting green fluorescent protein(GFP) and firefly luciferase(LUC) gene separately or both for the test of knock down property of these vectors.Results Constructed single and two site targeting RNAi vectors got desirable knockdown effects,in which single site targeting vector could get about 90 percent knock down efficiency whereas two site targeting vector reached about 87.5 percent knock down efficiency either.Conclusion Our RNAi vector construction strategy is efficient and time-saving so that it can be used for knocking down several genes simultaneously in the same cell.
ABSTRACT
Objective To construct the double-H1promoters siRNA expression cassettes(SECs) targeted to human telomere retrotranscriptase(hTERT),and investigate the interfering effect of its siRNA on hTERT gene expression in HepG2 cells.Methods SECs were constructed by fusing PCR,based on two different human telomerase hTERT gene fragments.When SEGs transferred into HepG2 cells respectively,the SECs were transcripted to the siRNA.The interfering effect of SECs on the telomerase activity in the cells was assessed by telomeric repeat amplification protocol(TRAP) and PCR-EIA.Results SEGs were successful constracted,and the telomerase activity was significantly inhibited when the HepG2 cells were tranfected with SECs.Conclusions The siRNA SECs display a definite RNA interference effect on the expression of telomerase.This method of SECs preparation can be applied for RNAi research in tumor inhibition.