GAL4-GFP Enhancer-trap lines
Donated by
- Jim Haseloff Department of Plant Sciences, University of Cambridge
Click here to view all 246 of these lines.
Description
Background
The GAL4-GFP lines were produced by Agrobacterium mediated transformation of the C24 Arabidopsis line using the plant binary vector pBIN m-gfp5-ER. This vector is a plant binary vector derived from pBI121 [1] in which the Bam HI-SstI fragment containing the Gus reporter gene has been replaced with a BamHI-SstI fragment containing the m-gfp5-ER gene [2&3. The modified m-gfp5-ER gene differs from the GFP gene originally isolated from the jellyfish Aequorea victoria in 4 critical ways which improve its expression in Arabidopsis
- Elimination of the cryptic intron which caused aberrant mRNA splicing in dicot plants [4].
- Fusion of endoplasmic reticulum [ER] retention signals which allows intracellular targeting of the GFP to the endoplasmic reticulum rather than to the cytoplasm and nucleoplasm [4].
- Mutation to produce improved folding of the modified GFP at higher temperatures [37°C] required to grow E.coli [thermotolerant folding] [5].
- Mutation to produce GFP with equalised excitation peaks at 395nm and 473nm - the GFP can be visualised with either long wavelength UV [e.g. hand-held lamp] or blue light [e.g. argon laser] [5&6].
The mgfp5-ER gene is GAL4 responsive and is linked to a GAL4 gene [a foreign transcription activator derived from the yeast GAL4 gene] in the GAL4-GFP lines described here. Thus patterns of GAL4 expression can be immediately and directly visualised by detection of GFP expression [green fluorescence within the endoplasmic reticulum of cells expressing the GAL4]. The GAL4-GFP enhancer trap screen which produced the plants listed here was designed to yield markers for the Arabidopsis root meristem. Of more than 7,500 transformants produced a collection of 250 Arabidopsis lines with distinct and stable patterns of GAL4 and GFP expression in the root were found. These lines provide a valuable set of markers where particular cell type are tagged and can be visualised in living plants.
Targeted mis-expression studies.
A chosen target gene can be cloned under control of a GAL4 responsive promoter, separately transformed into Arabidopsis and maintained silently in the absence of GAL4. Genetic crossing of this single line with any of the GAL4-GFP lines will allow specific activation of the target gene in particular tissue and cell types [as indicated by the presence of GFP fluorescence] and the phenotypic consequences of mis-expression, including those lethal to the organism, can be studied.
Note from Jim Haseloff
Some GAL4-GFP expression patterns can vary with different growth conditions or at different developmental stages. The lines contain a kanamycin resistance marker gene, and can also be screened directly for GFP fluorescence. We grow seedlings on GM plates [MS basal medium + Gamborg's vitamins + 1% sucrose], with 25mg/l kanamycin if required. The GFP marker is our mGFP5-ER localised variant, and the expression patterns have been documented using long wavelength UV epifluoresence microscopy and 488nm confocal microscopy. Please use the descriptions of GFP expression in the brindabella catalogue as a guide, not a rule and if you would like to contribute any new information or web link, contact me at the address below.
Jim Haseloff, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, UK. jh295@cam.ac.uk
Ordering lines
Four sets of GAL4-GFP lines are now available and fall into the following categories.
- Set 1 and 1a Lines pre-screened for root expression by Jim Haseloff and Sarah Hodge.
- Set KS Lines pre-screened for shoot and floral expression by Kirby Siemering and Marion Bauch.
- Set M Lines prescreened for shoot and floral expression by Marion Bauch.
| Nasc code | Description | Set contents |
|---|---|---|
| N9089 | GAL4-GFP enhancer trap complete set 1. Contains 116 lines (sets J and Q). | View set contents |
| N9340 | GAL4-GFP enhancer trap complete set 1a. Contains 30 lines. | View set contents |
| N9341 | GAL4-GFP enhancer trap complete set KS. Contains 32 lines. | View set contents |
| N9342 | GAL4-GFP enhancer trap complete set M. Contains 71 lines. | View set contents |
Related links
References
- Siemering, K.R., Golbik, R., Sever, R. & Haseloff, J. 1996. Mutations that suppress the thermosensitivity of green fluorescent protein. Current Biology 6(12):1653-63. PMID. 8994830.
- Zernicka-Goertz, M. et al. 1997. 1997. Following cell fate in the living mouse embryo. Development 124(6):1133-1137. PMID. 9102300.