Inverted repeat PCR for the rapid assembly of constructs to induce RNA interference.
Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel PCR strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs in any vector. IR-PCR relies on differentially tagging antisense and sense copies of the target in one round of PCR and assembling them in a second. We used IR-PCR to assemble constructs targeting profilin, actin, and actin-related protein (ARP) transcripts from Arabidopsis. Immunoblotting of lines expressing a profilin PRF1 3' untranslated region (UTR)-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.
Pawloski
Lucia Cardenas
LC
Department of Genetics, Life Sciences Building, University of Georgia, Athens, 30602, USA.
Deal
Roger B
RB
McKinney
Elizabeth C
EC
Burgos-Rivera
Brunilís
B
Meagher
Richard B
RB
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D020868:Gene Silencing; D016133:Polymerase Chain Reaction / Q000379:methods*; D034622:RNA Interference; D014158:Transcription, Genetic
D017931:DNA Primers