Biotechnology Letters

, Volume 30, Issue 7, pp 1271–1274 | Cite as

A modified T-vector for simplified assembly of hairpin RNAi constructs

Original Research Paper

Abstract

We describe a modified T-vector, pGFPm-T, for direct cloning of RT-PCR products to generate bidirectional restriction fragments for assembly of hairpin-containing RNAi vectors in the popular pFGC and pGSA binary vector backbone. Green fluorescence protein (GFP) is used as a visual reporter for direct selection of recombinants under UV illumination. The simplified cloning process enables a seamless workflow from candidate gene selection and RT-PCR verification to inverted repeat cloning, using a single pair of gene-specific primers.

Keywords

Cloning vector Dihydroflavonol reductase genes Gene silencing GFP Populus Transgenic plant 

References

  1. Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457–467PubMedCrossRefGoogle Scholar
  2. Kerschen A, Napoli CA, Jorgensen RA, Muller AE (2004) Effectiveness of RNA interference in transgenic plants. FEBS Lett 566:223–228PubMedCrossRefGoogle Scholar
  3. McGinnis K, Chandler V, Cone K, Kaeppler H, Kaeppler S, Kerschen A, Pikaard C, Richards E, Sidorenko L, Smith T, Springer N, Wulan T (2005) Transgene-induced RNA interference as a tool for plant functional genomics. Methods Enzymol 392:1–24PubMedCrossRefGoogle Scholar
  4. Mehta RK, Singh J (1999) Bridge-overlap-extension PCR method for constructing chimeric genes. BioTechniques 26:1082–1086PubMedGoogle Scholar
  5. Park KS, Park SW, Choi SY (2000) Improved T-vector for the cloning of PCR DNA using green fluorescent protein. J Microbiol Biotechnol 10:264–266Google Scholar
  6. Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529PubMedCrossRefGoogle Scholar
  7. Tsai CJ, Harding SA, Tschaplinski TJ, Lindroth RL, Yuan Y (2006) Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol 172:47–62PubMedCrossRefGoogle Scholar
  8. Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  9. Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in plants. Plant Physiol 141:1508–1518PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.Biotechnology Research Center, School of Forest Resources and Environmental ScienceMichigan Technological UniversityHoughtonUSA

Personalised recommendations