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Plant and Cell Physiology Advance Access originally published online on August 24, 2005
Plant and Cell Physiology 2005 46(11):1872-1878; doi:10.1093/pcp/pci191
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Short Communication

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

Lucia Cardenas Pawloski1, Roger B. Deal, Elizabeth C. McKinney, Brunilís Burgos-Rivera and Richard B. Meagher*

Department of Genetics, Life Sciences Building, University of Georgia, Athens, GA 30602, USA

* Corresponding author: E-mail, meagher{at}uga.edu; Fax, +1-706-542-1444.

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.

1 Present address: Center for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA.

(Received June 9, 2005; Accepted August 15, 2005)
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