Enhancer trapping in zebrafish using the Sleeping Beauty transposon - PubMed (original) (raw)

Enhancer trapping in zebrafish using the Sleeping Beauty transposon

Darius Balciunas et al. BMC Genomics. 2004.

Abstract

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.

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Figures

Figure 1

Artificial enhancer trapping with a Sleeping Beauty transposon. Comparison of GFP expression in embryos injected with pT2/

EF1α or γCry1/pT2/

EF1α. Plasmids are diagramed as cartoons on the top of the picture. The SB transposon's inverted repeats are shown as boxes with open triangles, and the GFP open reading frame is depicted as a grey arrow. The gamma-Crystallin promoter/enhancer is shown as a hatched box. DNA-injected embryos which survived to 3 dpf were counted and scored for GFP fluorescence anywhere in the embryo (any GFP) and for fluorescence in the eye (eye GFP), even if there was additional fluorescence elsewhere. The average percentage of embryos positive for particular GFP fluorescence in three independent experiments is shown ± standard deviation.

Figure 2

EF1α promoter truncations and endogenous enhancer trap screening. A diagram of the

EF1α promoter [32, 41]. Restriction enzyme sites are shown on top as single letters. S is _Sph_I, N is _Nhe_I, B is _Bst_1107I and R is _Eco_RI. G/C, G and C rich box. Sp1, Sp1-like site. TATA, TATA box. Numbering below is relative to the first T of the TATA box. The table below the diagram shows the results of the pilot and scale-up (*) screens. Transgenesis and expression rates are shown, non-expressing transposon insertions were not scored. Transgenesis and expression rate from scale-up screen (#) is an underestimate since many founders were screened by incross and crosses from doubly transgenic founders were scored as a single transmission event (see text).

Figure 3

Enhancer trap lines exhibit a variety of unique GFP expression patterns. (A). Lateral view of GFP expression in Enhancer Trap line 1 (ET1) at 38 hours post fertilization (hpf). (B) ET3 at 5–6 somite stage. (C) ET3 at 36 hpf. (D) ET4 at 26 hpf. (E) ET5 at 30 hpf. (F) ET5 at 48 hpf. (G) ET6 at 26 hpf. (H) ET7 at 32 hpf. (I) Ventral view of ET7 at 5 dpf. (J) Lateral view of ET8 at 26 hpf. (K) Dorsal view of ET9 at 28 hpf. (L) Lateral view of ET9 at 30 hpf. In all panels, anterior is to the left. See text for details.

Figure 4

The ET2 transgenic fish line expresses GFP in caudal primary motoneurons. GFP expression in ET2 was visualized in motoneurons using a bandpass GFP filter set at various stages of embryonic development. In all panels anterior is to the left. (A) The onset of GFP expression in ET2 line at 16 somite stage. (B) 26 somite stage. (C) 24 hpf. (D, E) 36 hpf. Axonal trajectories are visible at 24 and 36 hpf.

Figure 5

Identification of the transposition event in the ET2 line. (A) The pT2/

EF1α transposon insertion into zebrafish genome is shown; restriction enzyme sites and primers used for molecular analysis are indicated. Transposon IR/DR's are shown as solid boxes with open triangles, and the GFP open reading frame is shown as a grey arrow. Genomic DNA is shown as a dotted line. N is _Nsi_I, E is _Eco_RV. (B) Southern blot on ET2 line outcross embryos. DNA from GFP positive (lanes 1 and 2) and GFP negative (lanes 3 and 4) embryos was digested with _Nsi_I (lanes 1 and 3) or _Eco_RV (lanes 2 and 4) and probed with a GFP-specific probe. (C) Linkage of the transposon insertion event to GFP expression. Primers flanking the transposon insertion event (arrows) were used to conduct PCR on DNA from GFP positive (lane 2) and GFP negative (lane 3) embryos from an ET2 outcross different from the one used in (B). Lane 1, λ _Eco_47III Marker (Fermentas Inc).

Figure 6

GFP expression in ET2 line embryos is indistinguishable from endogenous PARG gene expression. 23 hpf embryos collected from a heterozygous outcross were photographed for GFP fluorescence and sibling embryos were fixed for in situ hybridization. (A) In situ hybridization with PARG antisense probe. (B) In situ with GFP antisense probe. (C) Visualization of GFP expression in living embryos using a bandpass GFP filter set. (D) The same embryo as in (C) photographed using a bandpass GFP filter set with a low level of bright field illumination to visualize GFP expression in relative position to the somites.

Figure 7

GFP expression in ET7 line matches mkp3 mRNA expression. (A) GFP fluorescence photograph of an ET7 embryo at 23 hpf. (B) In situ hybridization on 23 hpf wild type embryo using mkp3 antisense RNA probe.

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Enhancer trapping in zebrafish using the Sleeping Beauty transposon - PubMed (original) (raw)