Journal Article

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

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

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 11, pages 1872-1878
Published in print November 2005 | ISSN: 0032-0781
Published online November 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci191
Inverted Repeat PCR for the Rapid Assembly of Constructs to Induce RNA Interference

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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.

Keywords: Post-transcriptional gene silencing; RNAi; Stem-loop RNA; Transcriptional gene silencing; ARP, actin-related protein; GUS, β-glucuronidase; IR-PCR, inverted repeat PCR; NOS, nopaline synthase; PEPC, phosphoenolpyruvate carboxylase; PTGS, post-transcriptional gene silencing; RDRP, RNA-dependent RNA polymerase; RNAi, RNA interference; UTR, untranslated region; WT, wild type

Journal Article.  4668 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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