• Journal of Sericultural and Entomological Science
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• v.33 no.1
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• pp.21-26
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• 1991

Bombyx mori nuclear polyhedrosis virus (BmNPV) was successfully multiplied in the nuclear of BmN4 cells cultured with insect Grace's medium. By electron microscopic observation, the virons had a single nucleocapsid in an envelope. Polyhedral protein synthesis of BmNPV in BmN4 cells was detected at 18 hr p.i. and polyhedral protein was a singlepolypeptide with a M.W of 30 kd. At 48 hr p.i. polyhedra formation was observed by inverted mociroscope and electron microscope. Genome analysis of BmNPV by restriction endonucleases was not revealed the difference between virus produced in vivo and that in vitro.

• International Journal of Industrial Entomology and Biomaterials
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• v.40 no.1
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• pp.16-21
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• 2020

Humans use insects as food and traditional medicine for many years. Hemolymph is the circulating fluid of insects and is a key component of their immune system. However, limited information is available regarding hemolymph identification, development, and differentiation, as well as the related cellular immune responses. In a previous study, hemolymph extracts prepared from Bombyx mori larvae were found to exert anti-inflammatory effects. In this study, we aimed to identify and compare the antioxidant activity of immune-challenged and unchallenged B. mori hemolymph extracts in vitro. For this purpose, human epithelial Caco-2 cells were first exposed to oxidative stress and then treated with various concentrations and incubation times of either immune-challenged or unchallenged B. mori hemolymph extracts. Next, we determined the effect of treatment on the relative expression of GPX-1, SOD-1, and SOD-2 antioxidant marker genes. We found that the expression rates of the three marker genes were markedly higher at a immune-challenged hemolymph extract concentration of 80 ppm compared to those at other concentrations, and the antioxidant effects were enhanced after treatment for 48 hr. Thus, B. mori hemolymph extracts showed antioxidant activity within the limited time and dose. Especially, the immune-challenged B. mori hemolymph extracts showed higher the antioxidant activities than unchallenged one. The activity of silkworm hemolymph extracts could facilitate the development of new types of functional foods, feed additives, and biomaterials with antioxidant properties.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.1
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• pp.21-28
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• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2001.11a
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• pp.131-131
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• 2001

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2001.10a
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• pp.41-41
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• 2001

no Abstract, See Full Text

• Journal of Sericultural and Entomological Science
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• v.39 no.1
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• pp.36-43
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• 1997

In order to express the FLP recombinase in B. mori cultured cell line, BmN-4, transient expression system using a heat shock protein gene (hsp70) promoter of Dorosophilla melnogaster was constructed. This vector was designated as pHsSV. Activity strength of the hsp70 promoter was compared with that of immediate early gene (IE-1) and polyhedrin gene of BmNPV employing the E. coli $\beta$-galactosidase gene as a reporter gene. The result showed that the pHs $\beta$-gal plasmid vector expressed the $\beta$-galactosidase at 2nd and 3rd day after the transfer of plasmid DNA into BmN-4 cells, which was similar to that of pIE1 $\beta$-gal vector, but different from that of a recombinant virus, vBm $\beta$-gal. For the construction of FLP recombinase transient expression vector, the FLP recombinase gene was cloned by polymerase chain reaction technique. To express the FLP recombinase, this gene was inserted into pHsSV plasmid vector, under the control of the hsp70 promotor, and tranfected in BmN-4 cells. The expressed FLP recombinase was estimated at 44kDa on a 12.5% SDS-PAGE.

• International Journal of Industrial Entomology and Biomaterials
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• v.39 no.1
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• pp.14-21
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• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• International Journal of Industrial Entomology and Biomaterials
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• v.45 no.2
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• pp.99-107
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• 2022

Silkworm pupal extracts (SPE) were prepared in different solvents (water, 30%, 50%, 70%, and 100% ethanol) and their anti-inflammatory effects were evaluated in the RAW264.7 cell line. The SPE composition was initially evaluated by determining the protein content and performing Fourier transform infrared (FTIR) analysis. The protein content of the different SPE ranged from 6.75-130.93 mg/g of extract. FTIR analysis exhibited distinguishable absorption peaks among the extracts and indicated the presence of lipids, proteins, carbohydrates, and nucleic acid moieties. The levels of released nitric oxide (NO) and interleukin-6 (IL-6) expression in lipopolysaccharide (LPS)-induced RAW264.7 cells were only attenuated by 100% ethanolic SPE to 19.44% and 16.77%, respectively. The other solvent extracts were ineffective. Hence, further studies were conducted with 100% ethanolic SPE from three distinct stages of male and female silkworm pupae belonging to four silkworm varieties (Baegokjam; B, GoldenSilk; G, Juhwangjam; J, and YeonNokjam; Y). The best reduction in NO release and interleukin-1β (IL-1β) expression levels was achieved by the SPE of early female pupae belonging to the Baegokjam variety (32.72%) and those of early female pupae belonging to the Baegokjam and GoldenSilk (59.93%) varieties, respectively. The best reduction in IL-6 expression by 49.70% was achieved by SPE from female pupae of the mid-pupal stage belonging to the Baegokjam variety.

• Advances in Traditional Medicine
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• v.8 no.3
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• pp.228-235
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• 2008

This study was conducted to evaluate the estrogen activity of silkworm (Bombyx mori) pupa extracts and their fractions. Powdered samples of freeze-dried silkworm pupa were extracted at room temperature (RT), $40^{\circ}C$, $60^{\circ}C$, $80^{\circ}C$, and $100^{\circ}C$ in water (D.W), chloroform, ethyl acetate, and methanol for 6h and then filtered (0.45 um). The extracts were then freeze-dried. The estrogenic activity of these extracts was then investigated by competition binding assays using estrogen receptor ${\alpha}\;(ER{\alpha})$ and $ER{\beta}$, and by evaluating their effects on the proliferation of the human breast cancer cell line, MCF-7. Among the extracts evaluated, water extracts prepared at RT showed the highest binding affinity to $ER{\alpha}$ ($IC_{50}$, 1.76 ug/ml) and $ER{\beta}$ ($IC_{50}$, 0.07 ug/ml). In addition, MCF-7 cells that were treated with 62.5 ug/ml of the RT extract showed the greatest increase in proliferation (2-fold; 1291.79%) when compared to control cells (659.82%). Next, the water extract that was prepared at RT (sample 1) was dissolved in D.W. and further fractionated using a Dowex 50W - 8X ($H^+$) column. The flow-through and wash were then pooled together and freeze-dried (sample 2). The bound materials were then eluted with 20 mM NaCl, after which they were applied to a Dowex 1X2 - 200 ($Cl^-$) column and washed with D.W. to remove the sodium ions. The eluants were then freeze-dried (sample 3). Of these fractions, sample 2 showed the highest binding affinity to ER{\alpha} ($IC_{50}$, 1.44 ug/ml) and $ER{\beta}$ ($IC_{50}$, 1.18 ug/ml). In addition, MCF-7 cells that were treated with sample 2 (15.6 ug/ml) showed the largest increase in growth (1159.39%) when compared to control cells (525.26%). Taken together, these results suggest that the fraction of the RT water extract of silkworm pupa referred to as sample 2 may be useful as a phytoestrogen.

• Journal of Life Science
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• v.30 no.8
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• pp.701-707
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• 2020

Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and could be an effective way of shortening the time required.