Genomic Approaches: Enhancer Trap
In plants the widespread functional redundancy of most genes prevents the identification and analysis of loss-of-function mutants. Miss-expression and over-expression of these genes represents an alternative way to explore their function and since these effects are dominant they may be readily transferred other plant species. We are utilizing a transactivation system to indirectly miss/over-express random genes. Inducible expression of a foreign transcriptional activator drives miss/over-expression any gene adjacent to randomly inserted response elements, an approach that has already proved very successful in Drosophila. Lines from this inducible ‘misexpression population’ displaying dominant traits can be crossed into the background of a second ‘enhancer-trap population’ to restrict the trait to a specific pattern (Fig. 1).
Two transactivation systems designed for use in plants are currently being utilized by the project. We have generated a collection of 6000 enhancer trap lines utilizing the pBIN mGAL-mGFP5 HDEL #15 construct (Fig. 2a), produced and tested by Dr J. Haseloff (University of Cambridge, UK). Lines displaying GFP reporter expression are undergoing characterization of the expression pattern throughout development, using fluorescence and confocal microscopy (Figs. 3, 4, & 5).
The stability and consistency of these patterns of GFP expression is also being examined through successive generations to the T4. An alternative transactivation system (Fig 2f & 2g), designed and tested by Ian Moore (Oxford, UK) is also being utilized by the project. The system employs a GUS reporter and chimeric transactivation protein (LhG4) with an alternative DNA binding domain and recognition site. Misexpression populations for each of the transactivation systems are currently being produced by random insertion of the respective response elements (UAS or pOp6) in the background of an inducible transactivation source (GAL4 or LhGR). Data and images collected for each line in both the enhancer-trap and misexpression populations is being collected on a database (Fig 6) and will be made available shortly.