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Published on January 23, 2009; 10.1104/pp.108.133975

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Received December 12, 2008
Accepted January 20, 2009

Positive fluorescent selection permits precise, rapid and indepth over-expression analysis in plant protoplasts

Bastiaan Otto Robert Bargmann and Kenneth David Birnbaum *

Center for Genomics and Systems Biology, Biology Department, New York University, 100 Washington Square East, 1009 Silver Building, New York, NY 10003, USA

* Corresponding author; email: ken.birnbaum{at}nyu.edu.

Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the study of numerous aspects of plant biology. Recent studies in metazoan systems have utilized cell-based assays to interrogate signal transduction pathways using high-throughput methods. Plant biologists could benefit from new tools that expand the use of cell culture for large-scale analysis of gene function. We have developed a system that employs fluorescent positive selection in combination with flow cytometric analysis and fluorescence activated cell sorting (FACS) to isolate responses in the transformed protoplasts exclusively. The system overcomes the drawback that transfected protoplast suspensions are often a heterogeneous mix of cells that have and have not been successfully transformed. This Gateway-compatible system enables high-throughput screening of genetic circuitry using over-expression. The incorporation of a red fluorescent protein (RFP) selection marker enables combined utilization with widely available green fluorescent protein (GFP) tools. For instance, such a dual labeling approach allows cytometric analysis of GFP reporter gene activation expressly in the transformed cells or FACS-mediated isolation and downstream examination of over-expression effects in a specific GFP-marked cell population. Here, as an example, novel uses of this system are applied to the study of auxin signaling, exploiting the RFP/GFP dual labeling capability. In response to manipulation of the auxin-response network through over-expression of dominant negative auxin-signaling components, we quantify effects on DR5::GFP reporter gene activation as well as profile genome-wide transcriptional changes specifically in cells expressing a root epidermal marker.






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