RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of @entity1 genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting @entity1 and @entity19 @entity3546 , respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple @entity3546 siRNAs effectively silenced endogenous @entity3546 expression, inhibited @entity13020 -induced @entity3546 / @entity15914 reporter activity and expression of Wnt/ @entity3546 downstream genes. Silencing @entity3546 in mesenchymal stem cells inhibited @entity13020 -induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between @entity36265 and @entity13020 in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

A simplified and versatile system for the simultaneous expression of multiple siRNAs in XXXX cells using Gibson DNA Assembly.

@entity1 :: ('9606', 'Species') :: ['mammalian', 'human'] @entity19 :: ('10090', 'Species') :: ['mouse'] @entity3546 :: ('1499', 'Gene') :: ['b-catenin'] @entity13020 :: ('89780', 'Gene') :: ['Wnt3A'] @entity36265 :: ('2658', 'Gene') :: ['BMP9'] @entity15914 :: ('6925', 'Gene') :: ['Tcf4']
@entity1 :: (9606,Species) :: ['mammalian']
