The aim of this BBSRC funded project, a collaborative effort between the laboratories of Dr Mark Tester and Professor Julian Hibberd, was to generate a library of rice (Oryza sativa L. japonica cv. Nipponbare) enhancer trap lines expressing an exogenous transcription factor (gal4) and the green fluorescent protein (gfp) in specific cell-types and tissues.
Working closely with Dr Emmanuel Guiderdoni at the French scientific organization CIRAD (Centre de coopération internationale en recherche agronomique pour le développement) in Montpellier, France, we have produced more than 13,000 primary transformants harboring the gal4-gfp construct originally developed by Professor Jim Haseloff for enhancer trapping studies in Arabidopsis thaliana. We screened T0 adult plants (1,982 lines), T1 seed (2,684 lines) and T1 seedlings (2,667 lines) to identify lines with gfp expression specific to individual cell-types in roots, leaves and flowers. Expression of gfp in specific tissues (flower, leaf, seed, etc.) averaged 10% while expression frequency at the whole plant level was 29%. Click here for additional details regarding the construction and screening of our gal4-gfp library.
Genes of interest can be transactivated by placing them downstream of the upstream activating sequences element (UAS) of GAL4, and introducing these constructs into selected enhancer trap lines. Binding of GAL4 to the UAS element is what drives expression of GFP in the gal4-gfp enhancer trapping system and, thus, GAL4 should bind to the UAS sequence of these introduced constructs and activate expression of the gene of interest in the same pattern observed for GFP. We demonstrated in both calli and plants that GAL4 is capable of activating a UAS:GUS construct in patterns and intensities highly correlated with that of GFP, indicating that targeted expression or manipulation of any gene of interest is possible using this system.
This work was published: Johnson AAT, Hibberd JM, Gay C, Essa PA, Haseloff J, Tester, M, Guiderdoni E. Spatial control of transgene expression in rice (Oryza sativa L.) using the GAL4 enhancer trapping system. Volume 41, Issue 5, pages 779–789, March 2005.
Alex Johnson who made these lines is now at johnsa@unimelb.edu.au.