Establishment of transgenic lines for jumpstarter method using a composite transposon vector in the ladybird beetle, Harmonia axyridis.

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, Harmonia axyridis, 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 Drosophila. 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.

Transgenic tools for members of the genus Drosophila with sequenced genomes.

The sequencing of the genomes of 12 Drosophila species has created an opportunity for much in the way of comparative molecular analyses amongst these species. To aid that endeavor, we have made several transformation vectors based on the piggyBac transposon with 3xP3-EGFP and -ECFP transgenic markers that should be useful for mutagenesis and establishing the GAL4/UAS system in these species. We have tested the ability of mini-white to be used as a marker for insertional mutagenesis, and have observed mini-white derived pigmentation of the testes sheath in a subset of lines from D. pseudoobscura and D. virilis. We have incorporated a source of piggyBac transposase into nine Drosophila species, and have demonstrated the functionality of these transposase lines for mobilization of marked inserts in vivo. Additionally, we tested the ability of a D. melanogaster nanos enhancer element to drive expression of GAL4 in D. melanogaster, D. simulans, D. erecta, D. yakuba, D. pseudoobscura, and D. virilis. The efficacy of the nos-Gal4 transgene was determined by measuring the response of UAS-EGFPtub in all six species. Our results show that D. melanogaster nos-Gal4 drives expression in other species, to varying degrees, in similar spatiotemporal domains in the ovaries, testes, and embryos as seen in D. melanogaster. However, expression levels are variable, demonstrating the possible need to use species-specific promoters in some cases. In summary, we hope to provide a set of guidelines and basic tools, based upon this work, for both insertional mutagenesis and GAL4/UAS system-based experiments in multiple species of Drosophila.

A novel fusion protein that functions as an enhanced green fluorescent protein reporter and a tetracycline-controlled transcriptional activator.

Binary expression systems are widely employed to analyze gene function in vivo using transgenic organisms. The tetracycline-off (Tet-Off) system, which is a binary expression system that uses a tetracycline-controlled transactivator protein (tTA) and its tetracycline operator sequence (tetO) binding site, was developed as a method for temporally controlling gene expression. To facilitate the use of the Tet-Off system in animal species other than the model organisms that are widely used for genetic analysis, we constructed two different fusion proteins containing enhanced green fluorescent protein (EGFP) as the reporter gene and tTA as the transactivator, in different configurations. We assessed the utility of these fusion proteins designated as tTA-EGFP and EGFP-tTA in transgenic fruit flies. We showed that, although EGFP of both fusion proteins was efficiently fluoresced, transcriptional activation occurred only by the tTA-EGFP fusion protein. Furthermore, tetracycline (Tc) and doxycycline (Dox) both effectively inactivated tTA-EGFP, repressing gene expression under tetO control in a concentration-dependent manner. Additionally, the removal of Tc or Dox from the diet can recover the transactivator activity of tTA-EGFP in a concentration- and time-dependent manner. The tTA-EGFP fusion protein will therefore be useful in the analysis of gene function in a wide range of animal species.

Human protein factory for converting the transcriptome into an in vitro-expressed proteome,.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.