GAL4-GFP enhancer trap system.

A system for GAL4 targeted gene expression. An enhancer trap vector bearing a modified GAL4-VP16 gene was inserted randomly into the Arabidopsis genome by Agrobacterium mediated transformation. Cell specific activation of the GAL4-VP16 gene by cellular enhancer results in activation of a linked GFP gene, allowing simple characterisation of expression patterns. Targeted expression of another gene (X) can be induced by genetic crossing. For example, we can use the system to trigger cell ablation by targeted expression of a toxin.

Targeted misexpression technique
Adaptation for plants
GAL4-GFP enhancer trap
Modified GAL4-VP16 protein
Enhancer-trap screen
GAL4-UAS promoter
GAL4-based misexpression in plants
Catalogues of GAL4-GFP lines
Notes for use of catalogue
How to obtain the lines.
Transactivating genes by genetic crossing
Targeted cell ablation
Diphtheria toxin A chain
Fluorescent staining for dead cells
Exploring cell-cell interactions in the root meristem
Interaction between the lateral root cap and epidermis
Fluorescent tagging and ectopic expression

The GAL4 system
In order to genetically manipulate cells during meristem development, we have developed a scheme for targeted gene expression, which is based on a method widely used in Drosophila (Brand and Perrimon, Development 118:401-415, 1993). We have used an "enhancer-trap" strategy to generate many transgenic plants which express different patterns of a yeast transcription activator, GAL4. A chosen target gene can then be placed under the control of GAL4 upstream activation sequences (UAS), transformed into plants, and maintained silently in the absence of GAL4. Genetic crosses between this single line and any of the library of GAL4-containing lines specifically activates the target gene in a particular tissue or cell type. The phenotypic consequences of mis-expression, including those lethal to the organism, can be conveniently studied.We found that GAL4 is not expressed in Arabidopsis due to a high A/T content, which can interfere with mRNA processing in plants. We have altered the codon usage of a derivative, GAL4-VP16, so that it is expressed efficiently in plants, and randomly inserted the modified gene into the Arabidopsis genome, using Agrobacterium tumefaciens-mediated transformation. The transformation vector was designed so that expression of the GAL4-VP16 gene would be dependent upon the fortuitous proximity of an Arabidopsis enhancer element. The inserted DNA also contained a GAL4-responsive green fluorescent protein (GFP) gene. Thus, interesting "enhancer-trap" patterns of GAL4 gene expression were immediately and directly visible, with each GAL4-expressing cell marked by bright green fluorescence.

We have used in vivo detection of GFP to directly screen for GAL4-directed GFP expression in 7500 regenerated plantlets. We have documented a collection of 250 Arabidopsis lines with distinct and stable patterns of GAL4-VP16 and GFP expression in the root. These lines provide a valuable set of markers, where particular cell types are tagged and can be visualised with unprecedented ease and clarity in living plants. Most importantly, GAL4-VP16 expression within these lines can be used for targeted gene expression experiments. We have produced transgenic plants which maintain regulatory proteins or toxins, silent behind a GAL4-responsive promoter. We can now activate these genes in specific cells by crossing to a chosen GAL4-VP16 expressing line.