Dataset Information

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

ABSTRACT: In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

SUBMITTER: Roman A

PROVIDER: S-EPMC7217008 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

Román Ángela Á Golz John F JF Webb Alex A R AAR Graham Ian A IA Haydon Michael J MJ

The Plant journal : for cell and molecular biology 20191203 1

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circad ...[more]

PMID: 31692146

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Project description:BACKGROUND: Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study. RESULTS: A key feature of this study is the use of luciferase (LUC) as the primary reporter and rsGFP-GUS as secondary reporters. Reporters driven by a 5XUAS are better suited in Arabidopsis than those containing a 1X or 2X UAS. A 5XUAS-LUC reporter is expressed at high levels in Arabidopsis lines transformed with Gal4 driven by the full, enhanced 35S promoter. In contrast, a minimum 35S (containing the TATA region) upstream of Gal4 acts as an enhancer trap system. Luciferase expression in trap lines of the T1, T2, and T3 generations are generally stable but by the T4 generation approximately 25% of the lines are significantly silenced. This silencing is reversed by growing plants on media containing 5-aza-2'-deoxycytidine. Quantitative multiplex RT-PCR on the Gal4 and LUC mRNA indicate that this silencing can occur at the level of Gal4 or LUC transcription. Production of a 10,000 event library and observations on screening, along with the potential for a Gal4 driver system in other plant species are discussed. CONCLUSION: The Gal4 trap system described here uses the 5XUAS-LUC and 5XUAS rsGFP-GUS as reporters and allows for in planta quantitative screening, including the rapid monitoring for silencing. We conclude that in about 75% of the cases silencing is at the level of transcription of the Gal4 transgene and is at an acceptable frequency to make the Gal4 trap system in Arabidopsis of value. This system will be useful for the isolation and comprehensive characterization of specific reporter and driver lines.

Project description:BACKGROUND: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. DESCRIPTION: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. CONCLUSION: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

Dataset Information

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

Publications

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

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