RNA interference (RNAi) is a powerful tool for functional genomics in a number of species. The logistics and procedures for doing high-throughput RNAi to investigate the functions of large numbers of genes in Arabidopsis thaliana and in Zea mays are described. Publicly available plasmid vectors that facilitate the stable chromosomal integration of inverted repeat transgenes that trigger RNAi have been used to generate more than 50 independent transgenic lines each in Arabidopsis and maize. Analysis of mRNA abundance of the targeted genes in independent lines transformed with distinct constructs indicates that the success of RNAi-induced silencing is gene dependent. mRNA levels were not detectably reduced for some genes, but were dramatically reduced for a number of genes targeted. A common pattern was that multiple independent lines transgenic for the same construct showed the same extent of silencing. This chapter describes the procedures used to generate and test transgenic lines mediating RNAi in Arabidopsis and maize.
Bibliographical noteFunding Information:
This project was funded by a grant from the National Science Foundation (DBI-9975930) to Richard Jorgensen (principal investigator), Judith Bender, Vicki Chandler, Karen Cone, Stanton Gelvin, Heidi Kaeppler, Shawn Kaeppler, David Mount, Craig Pikaard, and Eric Richards (co-principal investigators). We thank Carolyn Napoli and Rich Jorgensen for vector design; Rayeann Archibald and Virginia O'Connell for vector construction; Judith Bender for participating in the generation and testing of Arabidopsis transgenic lines; Andreas Muller for supervising the Arabidopsis RT-PCR experiments, Ross Atkinson for generating and testing the inducible silencing vectors; Nives Kovacevic for assistance with maize T 1 analyses; and the following technicians, specialists, and students who contributed their efforts to this project—Heather Basinger, Dean Bergstrom, Erin Berry, Kari Hesselbach Chambers, Heather Ferguson, Miriam Hankins, William Haun, Kirsten Lovette, Jill Mahoy, Annie McGill, Justin Rincker, Robert Sandoval Jr., Laura Schmitt, Alan Smith, Kevin VerWeire, and Jessica Yoyokie.