Tol2-mediated transgenesis with pTol2{rps9::egfp} yields robust, ubiquitous expression of EGFP in G₀ animals.

<p>(<b>A, B</b>) pTol2{rps9::egfp}^frkt868-injected animals co-injected with <i>tol2</i> mRNA (A) show significantly stronger EGFP fluorescence (green arrows) than those without transposase (B) (ae: autofluorescence around the adult eyes). (<b>C, D</b>) Stable ubiquitous EGFP expression (green arrows) two months after co-injection of donor DNA and <i>tol2</i> mRNA (C) compared to a non-injected animal (D). Labels indicate autofluorescence of the jaws (j) as well as the iridophore pigments (p) in both the head and trunk.</p

Excision of a microinjected Mos-based construct.

<p>(<b>A</b>) Scheme of donor vector pMos{rps9::egfp}^frkt1074; in analogy to pTol2{rps9::egfp}^frkt868 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone-0093076-g001" target="_blank">Figure 1A</a>), the rps::egfp cassette is flanked by inverted Mos repeats (MosIR) that serve as recognition site for Mos1 transposase. (<b>B, C</b>) PCR using vector-specific primers (black arrows) yields a variety PCR fragment from embryos co-injected with <i>mos1</i> transposase mRNA (left lanes), but not controls (right lanes). (<b>D</b>) Sequencing of 21 individual PCR product reveals imprecise breakpoints caused by impaired excision of reporter constructs and/or subsequent modification of DNA ends in the context of non-homologous end repair. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone.0093076.s003" target="_blank">Alignments S1</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone.0093076.s004" target="_blank">S2</a></b> for details.</p

Efficiency of Tol2 and Mos1 transposon-mediated transient transgenesis.

<p>Efficiency is based on the detection of broad Egfp expression 7 days after injection of either pTol2{rps9::egfp}^frkt868 (left column) or pMos{rps9::egfp}^frkt1074 (right column). Percentages are calculated as the fraction of Egfp-expressing animals of total survivors for each of the constructs.</p

Tools for Gene-Regulatory Analyses in the Marine Annelid <i>Platynereis dumerilii</i>

<div><p>The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid <i>Platynereis</i>, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In <i>Platynereis</i>, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.</p></div

Genomic integration and inheritance of Tol2-mediated constructs.

<p>(<b>A</b>) Genomic Southern blot with an <i>egfp</i> probe reveals genomic integrations of pTol2{rps9::egfp}^frkt868 in tested G₀ individuals (arrows). (<b>B</b>) Precise genomic integration of a the reporter construct at the end of the IR as revealed by sequencing of an amplicon including neighboring genomic DNA, generated using Tol2IR-specific TAIL-PCR primers. (<b>C</b>) Integration and inheritance of <i>egfp</i> in individual G₀ and G₁ animals: PCR amplicons specific for <i>egfp</i> (arrowheads) can be detected in genomic DNA of both G₀ (lanes 9–18) and G₁ (lanes 1–8) individuals. Controls: no DNA (lane 19), wild-type gDNA (lane 20), gDNA extracted from a tuba::egfp^vbci1 transgenic individual (lane 21), reporter construct plasmid DNA (lane 22).</p

EGFP expression at the trochophore stage reflects endogenous <i>tuba</i> expression in tuba::egfp^vbci1 animals.

<p>(<b>A</b>) Scheme of donor vector pMos{tuba::egfp}^frkt707 containing 4.4 kB of DNA upstream of the <i>alpha-tubulin (tuba)</i> start codon (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone-0093076-g002" target="_blank">Figure 2A</a>). (<b>B, C</b>) Expression of endogenous <i>tuba</i> in the prototroch (pt; arrows), apical ciliary tuft (at) and paratrochs (pat; arrowheads) of <i>Platynereis</i> larvae at the trochophore (B) and metatrochophore (C) stages. (<b>D, E</b>) Expression of <i>egfp</i> RNA (D) and EGFP protein in tuba::egfp^vbci1 trochophore larvae perfectly reflects endogenous <i>tuba</i> expression. (<b>F</b>) <i>egfp</i> expression in tuba::egfp^vbci1 metatrochophore larvae remains restricted to the prototroch (arrow); (<b>G</b>) Stable inheritance of the construct, as evidenced by Southern blots of carriers from three different generations, probed with a labeled <i>egfp</i> fragment.</p

Survival and transmission rates for the injection of Mos1-based fluorescent reporter constructs.

<p>Data are shown for both pMos{tuba::egfp}^frkt707 and pMos{r-opsin::egfp}^frkt890 injections. Injected zygotes (left column) were raised to mature animals (G₀; middle column), and EGFP expression in the offspring (G₁; right column) was monitored. Survival rate and transgenic founder rate are given as percentage of injected animals and mature animals, respectively.</p

The <i>maf</i> locus drives EGFP in putative neurosecretory brain cells

<p>. (<b>A</b>) Scheme of donor vector pTol2{maf::egfp}^frkt1208 containing a 3.6 kB upstream of the <i>maf</i> start codon (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone-0093076-g001" target="_blank">Figure 1A</a>). (<b>B</b>) Expression of endogenous <i>maf</i> RNA (arrowhead) in the medial central brain of early metatrochophore larvae. (<b>C</b>) Synexpression of the <i>insulin-like peptide 2</i>/<i>ilp2</i> mRNA in the same region (arrowhead), suggesting that <i>maf</i> demarcates neurosecretory cells. (D–F) EGFP expression driven by pTol2{maf::egfp}^frkt1208 demarcates cells in the same region as endogenous <i>maf</i>.</p

Excision of a microinjected Tol2-based construct.

<p>(<b>A</b>) Scheme of donor vector pTol2{rps9::egfp}^frkt868; the rps::egfp cassette is flanked by inverted Tol2 repeats (Tol2IR) that serve as recognition site for Tol2 transposase co-injected as mRNA along with the vector DNA. (<b>B, C</b>) PCR using vector-specific primers (black arrows) yields a 200 bp PCR fragment specifically from embryos co-injected with <i>tol2</i> transposase mRNA (left lane), but not from controls (right lane). (<b>D</b>) Precise cleavage of the reporter construct at the end of the Tol2 IRs is evidenced by sequencing of the 200 bp fragment.</p

The <i>maf</i> locus drives EGFP in putative neurosecretory brain cells

<p>. (<b>A</b>) Scheme of donor vector pTol2{maf::egfp}^frkt1208 containing a 3.6 kB upstream of the <i>maf</i> start codon (compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093076#pone-0093076-g001" target="_blank">Figure 1A</a>). (<b>B</b>) Expression of endogenous <i>maf</i> RNA (arrowhead) in the medial central brain of early metatrochophore larvae. (<b>C</b>) Synexpression of the <i>insulin-like peptide 2</i>/<i>ilp2</i> mRNA in the same region (arrowhead), suggesting that <i>maf</i> demarcates neurosecretory cells. (D–F) EGFP expression driven by pTol2{maf::egfp}^frkt1208 demarcates cells in the same region as endogenous <i>maf</i>.</p