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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 4:457–467
Kerschen A, Napoli CA, Jorgensen RA, Muller AE (2004) Effectiveness of RNA interference in transgenic plants. FEBS Lett 566:223–228
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 392:1–24
Mehta RK, Singh J (1999) Bridge-overlap-extension PCR method for constructing chimeric genes. BioTechniques 26:1082–1086
Park KS, Park SW, Choi SY (2000) Improved T-vector for the cloning of PCR DNA using green fluorescent protein. J Microbiol Biotechnol 10:264–266
Tomari Y, Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19:517–529
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 172:47–62
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 27:581–590
Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in plants. Plant Physiol 141:1508–1518
Acknowledgments
We thank Dr. Soon-Yong Choi (HanNam University, South Korea) for providing the pHNT vector. This work was supported by the US Department of Energy, Office of Science, Biological and Environmental Research Program (DE-FG02-05ER64112), and the US National Science Foundation Plant Genome Program (DBI-0421756).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Luo, K., Harding, S.A. & Tsai, CJ. A modified T-vector for simplified assembly of hairpin RNAi constructs. Biotechnol Lett 30, 1271–1274 (2008). https://doi.org/10.1007/s10529-008-9673-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-008-9673-x