One-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system.

TitleOne-step cloning of intron-containing hairpin RNA constructs for RNA interference via isothermal in vitro recombination system.
Publication TypeJournal Article
Year of Publication2013
AuthorsJiang Y, Xie M, Zhu Q, Ma X, Li X, Liu Y, Zhang Q
JournalPlanta
Volume238
Issue2
Pagination325-30
Date Published2013 Aug
Abstract

Hairpin RNA-based RNA interference (hpRNAi) has become a powerful tool for exploring gene function in reverse genetics. Although, several methods are available for making constructs that express hpRNAi, multiple time-consuming cloning steps are usually involved. Here, we introduce an efficient and flexible hpRNAi vector construction method via the isothermal in vitro recombination system (IR-hpRNAi). For an IR-hpRNAi reaction, two PCR products of a target gene sequence are generated, which containS complementary ends (~20 bp) to each other and to the ends of linearized vector, are fused in a way of head-to-head or tail-to-tail into the vector. This IR-hpRNAi method offers two options to construct the RNAi vectors. Using this method, we created a IR-hpRNAi construct for the Arabidopsis PDS3 gene,and verified the silencing effect via Agrobacterium-mediated transformation. The IR-hpRNAi system rules out the requirement of engineering restriction enzyme cutting sites in target DNA fragments, and is ligation-independent. Thus, this method has advantages over the other hpRNAi construction methods.

DOI10.1007/s00264-013-1920-7
Alternate JournalPlanta