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Plant and Cell Physiology Advance Access published online on August 24, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci191
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received June 9, 2005
Accepted August 15, 2005

Short Communication

Inverted Repeat PCR for the Rapid Assembly of Constructs to Induce RNA Interference

Lucia Cardenas Pawloski 1, Roger B. Deal 2, Elizabeth C. McKinney 2, Brunilís Burgos-Rivera 2, and Richard B. Meagher 2*

1 Department of Genetics, Life Sciences Building, University of Georgia, Athens, GA 30602, USA; Current Address: Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333
2 Department of Genetics, Life Sciences Building, University of Georgia, Athens, GA 30602, USA

* To whom correspondence should be addressed.
Richard B. Meagher, E-mail: meagher{at}uga.edu


  Abstract

Expressing stem-loop RNAs in plants, fungi, and animals efficiently silences homologous target gene expression. We devised a novel polymerase chain reaction (PCR) strategy, called inverted repeat PCR (IR-PCR), which allows rapid assembly and cloning of stem-loop-containing constructs for gene silencing. 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'UTR-specific construct demonstrated a 77 to 97% reduction in PRF1 protein, but not other profilin isovariants.

Keywords: stem-loop RNA; post-transcriptional and transcriptional gene silencing; PTGS; TGS; RNAi.
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