A strategy for rapidly constructing the single and dual-site targeting plasmid-based RNAi vector / 基础医学与临床

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.