Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm pupa.

<p>(A–C) Two-day old pupa. EGFP expression was evident throughout the pupal body. (A, A’) Dorsal views under white light (A) and EGFP-excitation wavelength light (A’). Upper pupa is non-transgenic pupa. (B) Ventral view. (C) Lateral view. (D) A ventral view of the head and thorax of 4-day old pupa. DsRed expression was evident in the compound eyes, whereas EGFP was not. Abbreviations: an, antenna; ce, compound eye; sp, spiracle; wg, wing. Scale bar = 5 mm.</p

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in tissues dissected from 5th instar silkworm larvae.

<p>(A, A’) A dissected non-transgenic 5th instar larva. (B, B’) A dissected transgenic 5th instar larva. Intense EGFP fluorescence was evident in the anterior and posterior midgut. (C–J’) A dissected 5th instar larva. (C) Prothoracic gland. (D) Merged image of the transmitted light and EGFP fluorescence in the suboesophageal body. (E–F’) Trichogen (or trichogen and tormogen) cells in the epidermis in a non-transgenic larva (E, E’) and a transgenic larva (F, F’). (G–H’) Ovary of a non-transgenic larva (G, G’) and a transgenic larva (H, H’). EGFP was evident in tracheolar cells that were attached to the ovary. (I–J’) Tissues surrounding dorsal vessel of a non-transgenic larva (I, I’) and a transgenic larva (J, J’). EGFP was evident in pericardial cells along dorsal vessel and on the alary muscle, and peritracheal athrocytes, but not in fatbody. (A, B, E–J) White light, (A’, B’, C, E’-J’) EGFP-excitation wavelength light. The images for the comparisons of non-transgenic and transgenic larvae and tissues were obtained exactly by the same conditions. Abbreviations: dv, dorsal vessel; fb, fatbody; mg, midgut; pa, peritracheal athrocytes. Scale bars = 5 mm in (A) and (B), 500 µm in (C, E, F, G, H), 1 mm in (I, J).</p

A Baculovirus Immediate-Early Gene, <em>ie1</em>, Promoter Drives Efficient Expression of a Transgene in Both <em>Drosophila melanogaster</em> and <em>Bombyx mori</em>

<div><p>Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, <em>ie1</em>, from the <em>Bombyx mori</em> nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a <em>piggyBac</em>-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV <em>ie1</em> promoter; we performed a detailed investigation of the promoter in transgene expression pattern in <em>Drosophila melanogaster</em> and in <em>B. mori</em>. <em>Drosophila</em> and <em>Bombyx</em> belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, <em>ie1</em> promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the <em>ie1</em> promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous <em>ie1</em> promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the <em>ie1</em> promoter can be used as an efficient expression driver in a diverse range of insect species.</p> </div

Schematic representation of the <i>piggyBac</i>-based BmNPV <i>ie1</i> promoter reporter constructs.

<p>This construct was designated pBac[BmNPV <i>ie1</i>-EGFP, 3xP3-DsRed]. A fragment containing the sequences –631 to –2 bp upstream of the codon encoding the translational start site of BmNPV <i>ie1</i> was used as the BmNPV <i>ie1</i> promoter and to drive expression of EGFP. DsRed was under the control of 3xP3 and was used as the transformation marker. Abbreviations: ITR, inverted terminal repeats of <i>piggyBac</i>; hsp70 polyA, hsp70 polyadenylation signal; SV40 polyA, SV40 polyadenylation signal.</p

Genomic DNA sequences surrounding <i>piggyBac</i> insertions.

<p>The flanking sequences of <i>piggyBac</i> insertion in <i>D. melanogaster</i> and <i>B. mori</i> have 100% identity with the genome DNA sequences of chromosome 3L and Bm_scaf 21 in chromosome 17, respectively. Abbreviations: Dm, <i>Drosophila melanogaster</i>; Bm, <i>Bombyx mori</i>.</p

Expression pattern of the BmNPV <i>ie1</i>-EGFP transgene in silkworm larva at different stages.

<p>(A, A’) First instar larva just after hatching. BmNPV <i>ie1</i> promoter-driven EGFP expression did not overlap with 3xP3-driven DsRed expression in the ventral nerve cord. Ventral view. (B) Late 2nd instar larva. EGFP was expressed throughout the whole body and throughout all larval stages. Dorsal view. (C) Early 3rd instar larva. Dorsal view. (D, D’) 4th instar larvae. Dorsal view. Upper larva is a non-transgenic larva. (E) Head and thorax of 5th instar larva. Lateral view. Scale bars = 2 mm.</p

Results of inverse PCR for confirming remobilization of mutator.

<p>We performed inverse PCR using extracted DNA from original mutator individual and G₄ individuals. Each G₄ individual was derived from G₂ individuals (#2–5, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100804#pone-0100804-g003" target="_blank">Fig. 3E</a>). Each mutator inserted to different position in each genome.</p

Jumpstarter method in <i>Harmonia axyridis</i>.

<p>(A) Crossing scheme for jumpstarter method. A mutator line and a jumpstarter line are crossed (G₁). Newly hatched larvae expressing both ECFP and DsRed transformation markers in the CNS were selected from the progeny (G₂). Larvae carrying both the mutator and jumpstarter elements were heat shocked to induce <i>transposase</i> expression. Newly hatched larvae expressing only the DsRed transformation marker were selected from the progeny (G₃), and analyzed by PCR for mobilization of the mutator (i.e. integration elsewhere in the genome). Each unique insertion line was used to establish a new mutator line. (B) Schematic representation of PCR method to analyze mobilization of the original mutator element. Primer set HaG1 and PLR was used to detect the original non-mobilized mutator element. If the original mutator element is remobilized, no PCR product will be detected. Primer set HaG1-HaG3 was used as a positive control to detect the homologous, mutator element-free chromosome. (C) Red circles denote progeny with apparent remobilization events. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle, because the original mutator element was mobilized to other genomic sites. These larvae were established as new mutator lines. Each G₂ female gave at least one newly mobilized progeny. (D) Comparison of DsRed fluorescence between original mutator line (middle) and two new mutator lines (left and right). Compare to original mutator line (middle), two new mutator line larvae show higher (left) or lower (right) expression of DsRed. (E) PCR analysis of mutator element mobilization. Newly hatched G₃ larvae strongly or weakly expressing DsRed marker compared with original mutator line were selected and subjected to PCR analysis. PCR amplification with the PLR-HaG1 primer set was not detected in progeny marked with a red circle. In this case, detection efficiency of remobilized mutator element is increased compared with random selection (see result in C). (F) An enhancer-trap line of transgenic ladybird beetles. During the generation of mobilized mutator lines using the jumpstarter method, an enhancer expressing GFP throughout the body was detected (left); a wild-type control larva is also shown on the right. Panel displays a ventral view (anterior uppermost).</p

Schematic of composite vector and overview of genomic integration.

<p>(A) The left half of the composite vector contains a 5′ ITR, a <i>transposase</i> gene under the control of the <i>Dmhsp70</i> promoter and the 3xP3-<i>ECFP</i> transformation marker, while the right half contains both 5′ and 3′ ITRs, and is marked by both an <i>EGFP</i> gene under the control of the <i>Dmhsp27 </i>minimal promoter for enhancer trapping and a 3xP3-<i>DsRed</i> transformation marker. The <i>transposase</i> gene can be immobilized by the precise excision of the sequence between the central 5′ ITR and the 3′ ITR; in this case, larvae expressed ECFP alone. (B) Transformation markers expressed in the CNS of first instar larvae of <i>H. axyridis</i> indicate vector integration. From left to right, bright field, DsRed, ECFP and EGFP are presented in wild-type, composite vector (DsRed and ECFP are expressing), Jumpstarter (only ECFP is expressing) and Mutator (only DsRed is expressing). EGFP expression is not detected in all of strains. All panels display a ventral view (anterior uppermost).</p

Establishment of Transgenic Lines for Jumpstarter Method Using a Composite Transposon Vector in the Ladybird Beetle, <i>Harmonia axyridis</i>

<div><p>In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, <i>Harmonia axyridis</i>, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G₂ females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in <i>Drosophila</i>. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism.</p></div