Keiro Uchino

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Papers by Keiro Uchino

Fluorescence imaging for a noninvasive in vivo toxicity-test using a transgenic silkworm expressing green fluorescent protein

Scientific reports, 2015

In drug development, the toxicity of candidate chemicals must be carefully examined in an animal ... more In drug development, the toxicity of candidate chemicals must be carefully examined in an animal model. Here we developed a live imaging technique using silkworms for a noninvasive toxicity test applicable for drug screening. Injection of carbon tetrachloride, a tissue-injuring chemical, into transgenic silkworms expressing green fluorescent protein (GFP) induced leakage of GFP from the tissues into the hemolymph. The leakage of GFP was suppressed by pre-administration of either cimetidine, a cytochrome P450 inhibitor, or N-acetyl cysteine, a free-radical scavenger. The transgenic silkworm was made transparent by feeding a diet containing chemicals that inhibit uric acid deposition in the epithelial cells. In the transparent silkworms, GFP fluorescence in the fat body could be observed from outside the body. Injection of salicylic acid or iron sulfate, tissue-injuring chemicals, into the transparent silkworms decreased the fluorescence intensity of the GFP in the fat body. These fin...

A Hox Gene Antennapedia Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori

The Journal of biological chemistry, Jan 26, 2016

Hox genes play a pivotal role in the determination of anteroposterior axis specificity during bil... more Hox genes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hox genes can also function in terminally differentiated tissue of the lepidopteran Bombyx mori. In this species, Antennapedia (Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antp can regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR and in situ hybridization analyses, we demonstrate that misexpression of Antp in the posterior silk gland induced ectopic expression of major silk protein genes as sericin-3, fhxh4 and fhxh5. These genes are normally expressed specifically in the middle silk gland as is Antp. Therefore, the evidence strongly suggests that Antp activates these silk protein genes in the middle silk gland. The...

METHOD FOR PRODUCING PENTAMERIC CRP, PENTAMERIC CRP-PRODUCING TRANSGENIC SILKWORM AND METHOD FOR CONSTRUCTING SAME, DNA ENCODING CANINE MONOMERIC CRP AND EXPRESSION VECTOR CONTAINING THE DNA

TRANSGENIC SILKWORM CAPABLE OF PRODUCING ANTIBODY AND METHOD FOR PRODUCTION THEREOF

A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

Bioscience Biotechnology and Biochemistry, 2007

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain pro... more We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.

Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator, mouse Bcl-2-associated X protein (mouse Bax)

Insect Molecular Biology

ABSTRACT

Evaluating promoter sequences for trapping an enhancer activity in the silkworm Bombyx mori. Journal of Insect Biotechnology and Sericology

Extensive length polymorphism of the silk fibroin gene in the Japanese oak silkmoth, Antheraea yamamai

Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

Scientific Reports, 2015

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier f... more Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential.

Conservation of fibroin gene promoter function between the domesticated silkworm Bombyx mori and the wild silkmoth Antheraea yamamai

To analyze the functional conservation between the fibroin H chain gene promoters in insects of t... more To analyze the functional conservation between the fibroin H chain gene promoters in insects of the Bombycidae and Saturniidae, we constructed transgenic silkworms expressing yeast GAL4 under the control of the Bombyx mori or Antheraea yamamai fibroin H chain gene promoter (BmFibH-GAL4 and AyFib-GAL4) and examined their EGFP expression patterns after mating with a homozygous UAS-EGFP strain. Those silkworms carrying the BmFibH-GAL4 and UAS-EGFP constructs showed strict tissue- and stage-specific EGFP expression: expression was observed only in the posterior division of the silk gland after the third day of the fifth instar and then gradually increased as the larvae developed. Similarly, those silkworms carrying AyFib-GAL4 and UAS-EGFP also showed EGFP expression in the posterior division of the silk gland after the third day of the fifth instar; however, some strains expressed EGFP in other tissues as well. Thus, the basic function of the promoter is conserved between the two famili...

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Development of a new piggyBac vactor for generating transgenic silkworms using the kynurenine 3-mono oxygenase gene

Rescue of the Aojuku white-egg translucent (w-3ol) Bombyx mori mutant by transgenic expression of the wild-type Bmwh3 gene

Journal of Insect Biotechnology and Sericology

We examined the Bombyx mori (silkworm) white gene, Bmwh3, for its possible utility as a transgene... more We examined the Bombyx mori (silkworm) white gene, Bmwh3, for its possible utility as a transgenesis marker in the B. mori Aojuku white-egg translucent mutant (w-3ol) strain. To express wild-type Bmwh3 in the w-3ol strain, we used the GAL4/UAS system. The A3-GAL4 strain used drives expression of the transgene under the control of the UAS throughout all tissues at all stages. Whereas wild-type silkworms have dark brown eggs, opaque white larval integuments, and dark brown eyes, the w-3ol mutants have white eggs, translucent larval integuments, and light brown eyes. Transgenic expression of Bmwh3 in w-3ol mutant silkworms restored the morphology of the larval integument by increasing the uric acid content to near normal and partially restored the dark brown phenotype of the compound eye by slightly increasing the accumulation of ommochrome pigment. Eggs transgenically expressing Bmwh3 were brown/white mosaics. Thus, when linked to a strong promoter, Bmwh3 is likely to be a useful mark...

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Bombyx mori mutant by transgenic expression of the wild-type Bmwh3 gene

FigS1C

Supplementary Figure 1

by Takaaki Daimon and Keiro Uchino

FigS2

Construction and long term preservation of clonal transgenic silkworms using a parthenogenetic strain

by Keiro Uchino and Vyacheslav Klymenko

Journal of Insect Physiology, 2015

For the functional analysis of insect genes as well as for the production of recombinant proteins... more For the functional analysis of insect genes as well as for the production of recombinant proteins for biomedical use, clonal transgenic silkworms are very useful. We examined if they could be produced in the parthenogenetic strain that had been maintained for more than 40years as a female line in which embryogenesis is induced with nearly 100% efficiency by a heat shock treatment of unfertilized eggs. All individuals have identical female genotype. Silkworm transgenesis requires injection of the DNA constructs into the non-diapausing eggs at the preblastodermal stage of embryogenesis. Since our parthenogenetic silkworms produce diapausing eggs, diapause programing was eliminated by incubating ovaries of the parthenogenetic strain in standard male larvae. Chorionated eggs were dissected from the implants, activated by the heat shock treatment and injected with the transgene construct. Several transgenic individuals occurred in the daughter generation. Southern blotting analysis of two randomly chosen transgenic lines VTG1 and VTG14 revealed multiple transgene insertions. Insertions found in the parental females were transferred to the next generation without any changes in their sites and copy numbers, suggesting that transgenic silkworms can be maintained as clonal strains with homozygous transgenes. Cryopreservation was developed for the storage of precious genotypes. As shown for the VTG1 and VTG14 lines, larval ovaries can be stored in DMSO at the temperature of liquid nitrogen, transferred to Grace&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s medium during defrosting, and then implanted into larvae of either sex of the standard silkworm strains C146 and w1-pnd. Chorionated eggs, which developed in the implants, were dissected and activated by the heat shock to obtain females (nearly 100% efficiency) or by a cold shock to induce development to both sexes in 4% of the eggs. It was then possible to establish bisexual lines homozygous for the transgene.

Targeted disruption of a single sex pheromone receptor gene completely abolishes in vivo pheromone response in the silkmoth

Scientific reports, 2015

Male moths use species-specific sex pheromones to identify and orientate toward conspecific femal... more Male moths use species-specific sex pheromones to identify and orientate toward conspecific females. Odorant receptors (ORs) for sex pheromone substances have been identified as sex pheromone receptors in various moth species. However, direct in vivo evidence linking the functional role of these ORs with behavioural responses is lacking. In the silkmoth, Bombyx mori, female moths emit two sex pheromone components, bombykol and bombykal, but only bombykol elicits sexual behaviour in male moths. A sex pheromone receptor BmOR1 is specifically tuned to bombykol and is expressed in specialized olfactory receptor neurons (ORNs) in the pheromone sensitive long sensilla trichodea of male silkmoth antennae. Here, we show that disruption of the BmOR1 gene, mediated by transcription activator-like effector nucleases (TALENs), completely removes ORN sensitivity to bombykol and corresponding pheromone-source searching behaviour in male moths. Furthermore, transgenic rescue of BmOR1 restored norm...

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yonemura2013

Effect of ATG initiation codon context motifs on the efficiency of translation of mRNA derived from exogenous genes in the transgenic silkworm, Bombyx mori

SpringerPlus, 2014

The context sequence motif surrounding the ATG initiation codon influences mRNA translation effic... more The context sequence motif surrounding the ATG initiation codon influences mRNA translation efficiency and affects protein production; however, the optimal sequence differs among species. To determine the optimal sequence for production of recombinant proteins in a transgenic silkworm, we compared 14-nucleotide context motifs around the ATG (ATG-context) in 50 silkworm genes and found the following consensus: (A/T)AN(A/T)ATCAAAatgN. We were also able to define the least-common motif: CCN(C/G)CGN(C/T/G)(G/C/T)(T/G)atgC, which served as a negative control. To examine the regulatory role of these motifs in protein expression, we constructed reporter plasmids containing different ATG-context motifs together with either the luciferase gene or an enhanced green fluorescent protein (EGFP) gene. These constructs were then used for comparison of luciferase reporter activity and EGFP production in BmN4 cells in vitro as well as in transgenic silkworms in vivo. We detected 10-fold higher lucif...

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