Zebrafish Enhancer TRAP Transgenic Line Database ZETRAP 2.0

Introduction

Development of the highly efficient transposon-based transgenesis techniques expressing GFP or other fluorescent proteins (FP) provided unprecedented possibility for in vivo studies of developmental processes in the optically transparent zebrafish larvae. ¹ The first version of database of transgenic lines of zebrafish, ZETRAP, aimed to inform the scientific community about 27 transgenic lines that GFP expression patterns were documented by a compound microscope. It contained sequence inputs detailing the flanking genomic regions in these transgenics.^2,3 ZETRAP has several limitations, including insufficient resolution of imaging and lack of links to other databases. In time, more sophisticated databases better integrated with Web-based resources were developed for transgenics and mutant zebrafish lines, such as zTrap ⁴ and ZFISHBOOK (http://www.zfishbook.org/). Some of these databases were generated and maintained by a single laboratory (ZETRAP, zTrap). Others are developed as a decentralized virtual database compiling information about fish lines generated and maintained by different labs around the world, as exemplified by the Zebrafish Information Network (ZFIN) and the more recent database of insertional mutants, ZFISHBOOK.

Here, we introduce ZETRAP 2.0. In addition to a substantially expanded number of transgenic lines expressing cytosolic GFP that were generated by a “rejump” of transposon from the two independent genomic sites and GFP expression pattern in 3–5 dpf larvae documented by confocal imaging, it also contains a novel set of transgenics expressing the membrane-tethered version of photosensitizer protein, KillerRed. Where possible, we linked the genes found in close proximity to the transposon insertion sites to the zebrafish genome in Ensembl.

Database Features

The database has been assembled using Adobe Dreamweaver CS4 v. 10.0 (Adobe Systems, San Jose, CA). Unlike the old version, ZETRAP 2.0 is constructed in a more convenient format that allows continuous updates without taking it offline. Since the first version became live, it was updated with small improvements. Whereas the old ZETRAP contained description of 27 transgenic lines from a single primary screen, ZETRAP 2.0 contains description of 124 transgenics derived from six screens. The database contains a detailed scheme of an enhancer trap screen and the transposon constructs used. Each transgenic line was allocated a page containing confocal images of fluorescent larvae. A brief summary of the expression pattern of fluorescent protein, genomic DNA sequences flanking the insertion sites determined by TAIL-PCR, and whenever possible, genomic localization of inserts accompany each depicted transgenic line. The mapped inserts are linked to specific genes in Ensemble, which names are highlighted in the ZETRAP. In this way, they are also linked to all other databases, including ZFIN. The flanking sequences are in a simple text format, which makes it possible to “copy and paste” this information for DNA homology analysis by Blast. Retained features from an old version of this database that were modified in ZETRAP 2.0 include the introduction, a glossary section with links to related ET lines, worldwide distribution of these ET lines, materials, and methods.

Past experience demonstrated that the online format can effectively inform the community on the availability of these new research tools for developmental studies and provides an additional channel to improve the distribution of ET lines.

Discussion

The main aim of ZETRAP 2.0 is to consolidate and organize information about many novel ET lines generated in six screens conducted in this laboratory. In particular, generation of ET transgenics expressing membrane-tethered KillerRed complements the existing bioimaging capability generated with cytosolic GFP.^1,5 Although several hundred transgenics were generated in these six screens,^6,7 we were unable to maintain all of them. From visual analysis of these transgenic lines, we conclude that FP expression in the skin could be detected within the first day of development. We also observed maternally inherited FP in some of our transgenics. The majority of ET transgenics are displaying their characteristically FP expression pattern by 3 dpf, when most of our imaging was made. The database further highlights various tissue-specific FP expression patterns in our collections of transgenics. In some cases the expression pattern was simple enough to be shown in a single image. More complex patterns required multiple images showing anatomical structures at different focal planes.

It was shown recently that the keratin4 core promoter used serendipitously in our enhancer trap screens is compatible with many tissue-specific enhancers. It uniquely combines equally efficient interactivity with various enhancers and ability to find and reveal their activity in various tissues.^8,9 This successful combination explains the wide range of tissues and high level of FP expression in these transgenics, making them very sensitive in vivo tools for developmental studies. In fact, despite a simple design of the transgene construct, these lines remain in high demand and given the usefulness of these transgenics as research tools, attempts are still being made to establish which gene(s) expression is represented by the transgene in ET lines that are high on a list of demands and publications. ¹⁰ This process has been helped by a substantial improvement of knowledge about zebrafish genome presented in Ensembl (http://www.ensembl.org/Danio_rerio/Info/Index). Recently, we have started isolating specific cell lineages expressing FP from these transgenics using fluorescent cell sorter with high efficiency. These primary cell lines could be used for various purposes including comparative genomics, cell biology, and physiology (unpublished results).

The ZETRAP 2.0 website has been available via the Internet since August 2011 at [URL: http://plover.imcb.a-star.edu.sg/webpages/home.html]. This database contains only enhancer trap transgenics generated by our laboratory. The insertional mutant collection generated recently can be assessed in ZFISHBOOK (http://www.zfishbook.org/).