ZETRAP 2.0: Zebrafish Enhancer TRAP lines database
The Tol2 transposable element was utilized to design and perform effective enhancer trapping in zebrafish. The enhancer trap (ET) construct carries the EGFP gene controlled by a partial promoter of keratin4 (ker4) gene. Cytosolic EGFP (cytGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers.
First, a first collection of stable transgenic ET lines derived from microinjection of the modified transposon DNA and transposase RNA was established. These transgenics are shown in a cluster ET1 - ET37 in the ZETRAP 2.0. During this screen 29 transgenic lines that displayed various specific EGFP expression patterns in addition to basal expression from the modified ker4 promoter were isolated (Parinov et al., 2004). Whereas some of these transgenics generated a lot of interest (eg. ET4, ET20) some other lines were never requested. Given that maintenance of these lines is a burden some of the lines may be discontinued eventually and removed from the database.
Second, a property of the non-autonomous transposon-based system to be remobilized in presence of transposase delivered into an egg by mRNA microinjection was utilized. The two different ET lines (donor lines) with a single insertion (donor site) of Tol2 transposon-based ET construct were used for this purpose. Injection of transposase mRNA into transgenic embryos ET33 (two screens) led to remobilization of the transposon from the donor site into a new genomic location (clusters ET33-1B-ET33-K11 and ET33-mi2a-ET33-mi93B). Injection of transposase mRNA into transgenic embryos ET33-E20 led to remobilization of the transposon from the donor site into a new genomic location (cluster Gateways 2A-84B). During thise screens, 208 new stable transgenic ET lines with diverse EGFP expression patterns were isolated (Kondrychyn et al., 2009).
Third, the cyt-GFP in the ET construct was replaced by the membrane-tagged KillerRed (memKR) and a couple of dosen of transgenic lines expressing the red mem-KR at the membrane were generated (cluster memKR1-21). These transgenics provide useful tools for optogenetic maiming of cells in the CNS and heart by dose-dependent illumination (Teh et al., 2010).
Fourth, we initiated a tranposition of the mem-KR ET construct by injection of transposase mRNA into memKR15 and further expanded a number of such transgenics (cluster memKR15-1 - memKR15-22). Thus comparing to ZETRAP the new version of database (ZETRAP 2.0) represents transgenics from five additional screens.
Fifth, the insertional mutagenesis screen using the gene-breaking cassette was initiated and data are prepared for release.
The aim of this database is to connect the insertion sites (with the adjacent genes highlighted) with the expression patterns of fluorescent proteins in all ET lines, which are still available. The majority of ET transgenics are displaying their characteristic FP expression pattern by 3 dpf, when most of imaging was made.
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 present at different focal planes.
The sequences are in a simple text format, which makes it possible to "copy and paste" this information for DNA homology analysis by Blast.
Details of this work have been published in:
3. Teh C, Chudakov D, Poon KL, Mamedov IZ, Sek JY, Shidlovsky K, Lukyanov S, Korzh V. (2010) Optogenetic in vivo cell manipulation using KillerRed-expressing zebrafish transgenics. BMC Dev Biol, 10:110.
Screen, images, mapping and fish maintenance - Igor Kondrychyn, Cathleen Teh, Marta Garcia-Lecea, Kar-Lai Poon, Ben Choo, William Go, Melvin Sin, Wei-Chang Poh, Jun-Yan Sek, Deepa Chandra Segaran, Alexander Emelyanov, Sergey Parinov and personnel of the IMCB Fish Facility,
Database - Youxin Guan, Ben Choo, Ann Kang, Igor Kondrychyn; IT Support - Kok Wee Yap.