Enhancer trapping in zebrafish using the Sleeping Beauty transposon.
BACKGROUND
Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem.


RESULTS
We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline.


CONCLUSIONS
This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.
Balciunas
Darius
D
Arnold and Mabel Beckman Center for Transposon Research, Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St SE, Minneapolis, MN-55455, USA. balci001@umn.edu
Davidson
Ann E
AE
Sivasubbu
Sridhar
S
Hermanson
Spencer B
SB
Welle
Zachary
Z
Ekker
Stephen C
SC
D000818:Animals; D004251:DNA Transposable Elements; D047108:Embryonic Development / Q000235:genetics; D004742:Enhancer Elements, Genetic; D018014:Gene Transfer Techniques; D023281:Genomics / Q000379:methods*; D005854:Germ Cells; D006026:Glycoside Hydrolases / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D017403:In Situ Hybridization; D009046:Motor Neurons / Q000378:metabolism; D010865:Pilot Projects; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase