Egg Cooling After Oviposition Extends the Permissive Period for Microinjection-Mediated Genome Modification in .

In general, transgenesis efficiency is largely dependent on the developmental status of eggs for microinjection. We investigated whether the relationship between transgenesis efficiency and cooling eggs in silkworms, , affects the transgenesis frequencies. First, we performed a microinjection using eggs of different developmental statuses at 25 °C.

An Effective Chemical Permeabilization of Silkworm Embryos.

The lipid layer surrounding the vitelline membrane of insect eggs has a critical role in the waterproofing and desiccation resistance of embryos. However, this lipid layer also prevents the flux of chemicals into the embryos, such as cryoprotectants, which are required for successful cryopreservation. The permeabilization studies of silkworm embryos remain insufficient.

Abolition of egg diapause by ablation of suboesophageal ganglion in parental females is compatible with genetic engineering methods.

Microinjection of genetic material into non-diapause eggs is required for genetic engineering of silkworms. Besides diapause could be useful for maintaining transgenic lines, a drawback of this technology is that most standard silkworm strains and experimental lines of interest produce diapausing eggs. Several approaches have been developed to abolish diapause but none are very efficient.

Temporal analysis of N-acetylglucosamine extension of N-glycans in the middle silk gland of silkworm Bombyx mori.

N-glycosylation of proteins is an important post-translational modification in eukaryotic cells. One of the key modifications in protein N-glycosylation is N-acetylglucosamine (GlcNAc) extension mediated by N-acetylglucosaminyltransferase I (GNTI), which triggers N-glycan maturation from high-mannose-type to hybrid- and complex-type structures in Golgi. However, the temporal contributions of GNTI to GlcNAc extension and the resultant N-glycan structures in insects have not been analyzed.

The red egg gene as a novel effective egg color marker for silkworm transgenesis.

Ommochromes are major pigments involved in coloration of eggs, eyes, and epidermis of arthropods. The recessive homozygous of egg and eye color mutant of Bombyx mori, red egg (re), exhibits red eggs and dark red eyes instead of normal purplish-brown eggs and black eyes, due to a defect in ommochrome pigment synthesis. The gene responsible for the re mutant is a major facilitator superfamily transporter gene, Bm-re.

Effect of chemical dechorionation on silkworm embryo viability.

The chorion covering/protecting insect egg, which has some effective functions such as providing mechanical strength, protecting eggs from external environments, and keeping moisture adjustment, is one of the principal barriers to manipulation, cryopreservation, and study of insect embryos. Here we evaluated the silkworm embryo viability after dechorionation using chemical reagents. We have developed an easy and effective method for chemical dechorionation that enables embryos to develop in culture, so that the larvae could normally grow.

Production of cloned transgenic silkworms by breeding non-diapausing parthenogenetic strains.

Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low.

[Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms].

　We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins.

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus.

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date.

Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: application to behavioral and developmental studies.

Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited.

Precise genome editing in the silkworm Bombyx mori using TALENs and ds- and ssDNA donors - A practical approach.

Engineered nucleases are able to introduce double stranded breaks at desired genomic locations. The breaks can be repaired by an error-prone non-homologous end joining (NHEJ) mechanism, or the repair process can be exploited to introduce precise DNA modifications by homology-directed repair (HDR) when provided with a suitable donor template. We designed a series of DNA donors including long dsDNA plasmids as well as short ssDNA oligonucleotides and compared the effectiveness of their utilization during gene targeting with highly efficient transcription activator-like effector nucleases (TALENs).

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene.

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

Hoxgenes 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 Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland.

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

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.

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

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.

Cell proliferation by silk gut incorporating FGF-2 protein microcrystals.

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.

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

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.

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

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.

Odorant concentration differentiator for intermittent olfactory signals.

Animals need to discriminate differences in spatiotemporally distributed sensory signals in terms of quality as well as quantity for generating adaptive behavior. Olfactory signals characterized by odor identity and concentration are intermittently distributed in the environment. From these intervals of stimulation, animals process odorant concentration to localize partners or food sources.

Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor.

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori.

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms.

Establishment of tools for neurogenetic analysis of sexual behavior in the silkmoth, Bombyx mori.

Background: Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking.

Results: In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.

Identification of a novel strong and ubiquitous promoter/enhancer in the silkworm Bombyx mori.

Transgenic techniques offer a valuable tool for determining gene functions. Although various promoters are available for use in gene overexpression, gene knockdown, and identification of transgenic individuals, there is nevertheless a lack of versatile promoters for such studies, and this dearth acts as a bottleneck, especially with regard to nonmodel organisms. Here, we succeeded in identifying a novel strong and ubiquitous promoter/enhancer in the silkworm.