Growth and Vegetative Development
Bargmann, Bastiaan , Spyropoulou, Eleni , Deveau-Rosen, Jason , Birnbaum, Kenneth .
Developing a high-throughput, transient, cell-type-specific silencing system in Arabidopsis root protoplasts.
Previously, our lab has utilized Fluorescence Activated Cell Sorting (FACS) of protoplasted Arabidopsis (Arabidopsis thaliana) roots bearing cell-type-specific GFP markers to achieve genome-wide, tissue-specific analysis of transcription in this organ. The analysis resulted in the identification of gene-clusters that are specifically expressed in particular cell-types of the root and yielded candidate cell-type-specific transcription factors that might be involved in the acquisition, regulation or maintenance of cell-specification. We have now devised a method to assess the transcriptional effects induced by the knock-down of such factors in a high-throughput fashion. Using transient transformation of cell-type-specific GFP-marked root protoplasts with a Gateway-compatible silencing vector containing an mRFP positive selection marker followed by dual-label-FACS, candidate transcription factors will be targeted and their downstream effects will be profiled by using microarrays. As a proof-of-principle approach we are targeting the WEREWOLF transcription factor in the epidermis, involved in atrichoblast-cell-fate determination, for which a number of downstream components have already been established and verified experimentally. We can demonstrate by means of microarray analysis of preserved protoplasts that specialized cell-types can retain their cell-specific transcriptional identity over several days, an important prerequisite for the success of this strategy as we would like to follow the effect of knock-down of the candidate transcription factors through time. In future, targeting of multiple (redundant) transcription factors at once and over-expression analyses are envisaged.
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1 - New York University, Center for Comparative Functional Genomics, 100 Washington Square East, 1009 Silver Building, New York, NY, 10003, USA
2 - New York University, Center for Comparative Functional Genomics
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM