Bombyx Mori Cell Line Research Articles

MicroRNA let-7 targets BmCDK1 to regulate cell proliferation and endomitosis of silk gland in the silkworm, Bombyx mori.

MicroRNAs play critical roles in multiple developmental processes in insects. Our previous study showed that CRISPR/Cas9-mediated knock down of the microRNA let-7 in silkworms increased the size of larvae and silk glands, thereby improving the silk production capacity. In this study, we elucidate the molecular mechanism underlying of let-7 regulates growth. Identification of differentially expressed genes in response to let-7 knock down revealed enrichment of pathways associated with cell proliferation and DNA replication. let-7 dysregulation affected the cell cycle and proliferation of the Bombyx mori cell line BmN. Dual-luciferase and target site mutation assays showed that BmCDK1 is a direct target gene of let-7, with only 1 binding site on its 3'-untranslated region. RNA interference of BmCDK1 inhibited cell proliferation, but this effect was counteracted by co-transfection with let-7 antagomir. Moreover, let-7 knock down induced BmCDK1 expression and promoted cell proliferation in multiple tissues, and further induced endomitosis in the silk gland in vivo. Knock down of BmCDK1 resulted in abnormal formation of a new epidermis, and larval development was arrested at the 2nd or 3rd molt stage. Taken together, our results demonstrated that BmCDK1 is a novel target of let-7 in cell fate determination, possessing potential for improving silk yield in silkworm.

Synthesis of 1,4-benzodioxan derivatives and the evaluation of their biological activity as a novel juvenile hormone signaling inhibitor.

Juvenile hormone (JH) signaling inhibitors may be used as insect growth regulators because of their ability to control metamorphosis and reproduction in insects by regulating the action of JH. We identified ethyl (E)-3-(4-{[7- (4-methoxycarbonylbenzyloxy)-1,4-benzodioxan-6-yl]methyl}phenyl)prop-2-enoate (EMBP) and observed its strong precocious metamorphosis-inducing activity against silkworm larvae. To further elucidate its mechanism of action, we investigated the effect of EMBP on the JH-mediated signaling pathway in vitro and in vivo. In a reporter assay using a Bombyx mori cell line, EMBP strongly suppressed the induction of reporter gene expression by Juvenile hormone I (JH I) in a concentration-dependent manner. A parallel rightward shift was observed in the dose-response curve of JH I after treatment with EMBP, indicating that EMBP competitively inhibited JH. Moreover, we monitored developmental changes in the JH-responsive gene, Krüppel homolog 1 (Kr-h1), and ecdysone-responsive gene, Broad-Complex (BRC), in EMBP-treated silkworm larvae. EMBP suppressed only the expression of Kr-h1 in third-instar larvae. Our results demonstrated that EMBP specifically regulates the JH-mediated Kr-h1 signaling pathway. EMBP could be used as a lead compound in the development of new insect growth regulators. © 2023 Society of Chemical Industry.

Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines.

Simple SummaryBombyx mori is a lepidopteran insect with economic value. Its genetic background is clear, and genome sequence is relatively complete, but the function of many genes has not been determined. The genetic code expansion system has become an important means of gene function research. In this study, a genetic code expansion system suitable for B. mori cells was established. This system included a modified tRNAPyl/Pyrrolysyl-tRNA synthetase (PylRS) pair from Methanosarcina mazei, the reporter gene D[TAG]G formed by DsRed and EGFP through amber stop codon TAG connection and the unnatural amino acid H-Lys(Boc)-OH. In silkworm BmE and BmNs cell lines, the reporter gene expression was strictly controlled by H-Lys(Boc)-OH in the presence of both PylRS and tRNAPyl. The silkworm genetic code expansion system established here is another useful controllable gene expression system besides tetracycline induced expression system.Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have established a genetic code expansion system for B. mori based on the well-researched tRNAPyl/PylRS pair from Methanosarcina mazei. We used H-Lys(Boc)-OH, which is a lysine derivative to efficiently and tightly control the expression of the reporter gene DsRed[TAG]EGFP (D[TAG]G), which encoded a H-Lys(Boc)-OH-bearing protein fused with DsRed and EGFP (here regarded as D[Boc]G) in B. mori cell lines BmE and BmNs. In D[TAG]G, the amber stop codon is recognized as the orthogonal tRNAPyl. Successful application of genetic code expansion system in silkworm cell lines will support the research into the function of silkworm genes and paves the way for the identification of new genes and protein markers in silkworm.

Identification of novel juvenile-hormone signaling activators via high-throughput screening with a chemical library.

Juvenile hormone (JH) is an insect-specific hormone that regulates molting and metamorphosis. Hence, JH signaling inhibitors (JHSIs) and activators (JHSAs) can be used as effective insect growth regulators (IGRs) for pest management. In our previous study, we established a high-throughput screening (HTS) system for exploration of novel JHSIs and JHSAs using a Bombyx mori cell line (BmN_JF&AR cells) and succeeded in identifying novel JHSIs from a chemical library. Here, we searched for novel JHSAs using this system. The four-step HTS yielded 10 compounds as candidate JHSAs; some of these compounds showed novel basic structures, whereas the others were composed of a 4-phenoxyphenoxymethyl skeleton, the basic structure of several existing JH analogs (pyriproxyfen and fenoxycarb). Topical application of seven compounds to B. mori larvae significantly prolonged the larval period, suggesting that the identified JHSAs may be promising IGRs targeting the JH signaling pathway.

Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library

Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

Proteotranscriptomics assisted gene annotation and spatial proteomics of Bombyx mori BmN4 cell line

BackgroundThe process of identifying all coding regions in a genome is crucial for any study at the level of molecular biology, ranging from single-gene cloning to genome-wide measurements using RNA-seq or mass spectrometry. While satisfactory annotation has been made feasible for well-studied model organisms through great efforts of big consortia, for most systems this kind of data is either absent or not adequately precise.ResultsCombining in-depth transcriptome sequencing and high resolution mass spectrometry, we here use proteotranscriptomics to improve gene annotation of protein-coding genes in the Bombyx mori cell line BmN4 which is an increasingly used tool for the analysis of piRNA biogenesis and function. Using this approach we provide the exact coding sequence and evidence for more than 6200 genes on the protein level. Furthermore using spatial proteomics, we establish the subcellular localization of thousands of these proteins. We show that our approach outperforms current Bombyx mori annotation attempts in terms of accuracy and coverage.ConclusionsWe show that proteotranscriptomics is an efficient, cost-effective and accurate approach to improve previous annotations or generate new gene models. As this technique is based on de-novo transcriptome assembly, it provides the possibility to study any species also in the absence of genome sequence information for which proteogenomics would be impossible.

Inhibition of silkworm vacuolar-type ATPase activity by its inhibitor Bafilomycin A1 induces caspase-dependent apoptosis in an embryonic cell line of silkworm.

Vacuolar-type ATPase (V-ATPase) is a type of hydrogen ion transporter located in the vesicular membrane-like system, which mediates active transport and intracellular acidification in various compartments. In mammals, V-ATPase has been reported to play a key role in cell proliferation and apoptosis. The studies of V-ATPase in silkworm mainly focus on the acidification regulation of midgut and silk gland and immune resistance. However, there are few reports about the function of silkworm V-ATPase on cell proliferation, autophagy, and apoptosis. Thus, the function of V-ATPase in a cell line of Bombyx mori (BmE) was investigated by treating the cell line with bafilomycin A1, a specific inhibitor of V-ATPase. Cell counting kit 8 (CCK8) and flow cytometry analysis showed that bafilomycin A1 treatment decreased the cell proliferation activity, affected the cell cycle progression and induced cell apoptosis. LysoTracker Red staining showed that the target of bafilomycin A1 is lysosome. The expression of all autophagy-related genes ( BmATG5, BmATG6, and BmATG8) decreased, indicating that cell autophagy was inhibited. The analysis of the apoptosis pathway demonstrated that inhibiting the activity of V-ATPase of BmE cells could promote mitochondria to release cytochrome C, inhibit the expression of BmIAP, and activate the caspase cascade to induce apoptosis. All these findings systematically illustrate the effects of V-ATPase on the proliferation, autophagy, and apoptosis in BmE cells, and provide new ideas and a theoretical basis for further study on the function of V-ATPase in BmE.

Construction of a Bombyx mori cell line that stably express the susceptible gene +nsd-2 of Bombyx mori bidensovirus.

Bombyx mori bidensovirus (BmBDV) is the only type species established in the new genusBidensovirus within the new family Bidnaviridae. The BmBDV infected silkworm midgut shows symptoms such as the nucleus of midgut columnar cells expanded. It is confirmed theBmBDV-sensitive gene +nsd-2, which encoding an amino acid transporter protein might be the receptor of BmBDV. The deletion of this gene causes the silkworm resistance to BmBDV infection. There is no permissive cell line for BmBDV infection has been established yet. The cell lines (BmN, etc.) are used broadly for silkworm viruses study are mainly resistant to BmBDV infection. Therefore, the establishment of BmBDV permissive cell line is particularly important for studying BmBDV in vitro. In this study, we successfully constructed a cell line BmN (+nsd-2), which expressed +nsd-2 gene stably. The NSD-2 expression and localization could be detected in BmN (+nsd-2 ) cells via RT-PCR, western blot and immunofluorescence. The interaction between NSD-2 and the major structural protein VP of BmBDV was determined by Co-IP. However, we failed to detect the replication of BmBDV. This cell lines constructed has the potential for identifying the protein binding to the NSD-2 and provide a basis for study of BmBDV co-receptors and invasion mechanism.

A comparative study of the cytotoxic effect of chlorogenic acid on bm-12 cells from bombyxmori (insect) and mammalian cell line

A comparative study of the cytotoxic effect of chlorogenic acid on bm-12 cells from bombyxmori (insect) and mammalian cell line

Suppression of Bm-Caspase-1 Expression in BmN Cells Enhances Recombinant Protein Production in a Baculovirus Expression Vector System

Baculovirus expression vector systems (BEVSs) have been widely used for recombinant protein production. Many studies have tried to improve the recombinant protein production by either modification of BEVSs vectors or by cell line selection. In this study, using a modified shRNA vector, we established a stable Bombyx mori cell line that significantly inhibits expression of caspase-1 after selection. We further compared cell proliferation and viability between caspase-1-suppressed cells and control cells, and found that there is no significant difference in these stable cell lines. We utilized these cell lines to analyze recombinant protein production after infection with recombinant baculovirus. We found that both intracellular and extracellular recombinant protein production significantly increased when expression of caspase-1 is inhibited in BmN cells. These data indicate that blocking the apoptotic pathway is a promising way to enhance recombinant protein production in BEVSs.

Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.

The Transcription Express Characteristics of Several Genes in the Process of <i>Bombyx mori</i> Ovarian Carcinoma

Bombyx mori cell line (BmN) comes fromBombyx moriovary cell subculture. In order to study the change of several genes transcription in the process ofBombyx moriovary cells primary culture and subculture, we usedBombyx moriovary organizations and BmN cell lines as research materials, used Real Time fluorescent quantitative RT-PCR to detect cyclin gene family (CyclinA, CyclinB, CyclinB3, CyclinE, CyclinL1), p53 and Telomerase genes transcription level in the ovary and BmN cell lines, and took Actin3 gene as reference to dispose the results. The results showed that in theBombyx moriBmN cell lines the expression of CyclinA, CyclinB, CyclinB3, CyclinE, CyclinL1 and Telomerase genes were higher than those in the ovary. The expression of CyclinB in the BmN was more then 3.8 which was 76 times higher than that in the ovary; The expression of p53 gene in the BmN cell was lower than that in the ovary; The expression of Telomerase gene in the BmN cell was higher than that in the ovary. The results accumulated a reliable data for further study on the the role of cyclin gene family, p53 gene, and Telomerase gene in the process ofBombyx moriovarian carcinoma.

Development and characterization of a new Bombyx mori cell line for protein expression

A Bombyx mori continuous cell line, designated DZNU-Bm-17, was established from larval ovaries. The cells were initially grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum and 3% heat inactivated B. mori hemolymph at 25±1°C and later adapted gradually to TNM-FH medium. Partially adhered refractive cells were the predominant cell type in the culture. The cells took about 1055days to complete 100 passages in TNM-FH medium. The population doubling time of the cell line was about 30–34h at 25±1°C. The cell population was largely diploid, but a few triploids and tetraploids were also observed. DNA profiles using simple sequence repeat loci established the differences between the DZNU-Bm-1, Bm-5, DZNU-Bm-12, DZNU-Bm-17, and BmN cell lines. The cell line was susceptible to budded virus of B. mori nucleopolyhedrovirus (BmNPV), and 85–92% of the cells harbored BmNPV with an average of 15 occlusion bodies/infected cell. The cells expressed the luciferase and green fluorescent proteins using the BmNPV bacmid vector. We suggest the usefulness of the DZNU-Bm-17 cell line for BmNPV-based baculoviral expression studies.

Quantitative analysis of three commonly-used insect cell-specific promoters’ activities by transient and baculovirus-mediated expression

The activities of three insect cell-specific promoters were compared and analyzed here, including the cytoplasmic Actin 3 (A3) promoter from Bombox mori (silkworm), immediately early 2 (ie2) promoter from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and the very late polyhedrin (polh) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Results of transient expression assays showed the activity of ie2 promoter was approximately forty fold higher in Spodoptera frugiperda 9 (Sf9) cells and thirty fold higher in ovarian cell line of Bombyx mori (BmN) than that of A3 promoter. However, it showed reverse effect in baculovirus system that the activity of A3 promoter became approximately ten fold higher than that of ie2 promoter in the late stage of baculovirus infection. We constructed recombinant baculoviruses with enhanced green fluorescent protein gene (EGFP) reporter gene controlled by individual A3, ie2 and polh promoter. The analysis result of EGFP expression level showed the activity of polh promoter was nearly 50 fold higher than that of ie2, and 5 times higher than that of A3 at 72 h post infection in Sf9 cells. The systemic studies on the activities of the three promoters indicate they are ideal drivers for the construction of a time sequential baculovirus expression system. Key words: Promoter activity, immediately early promoter, very late promoter, transient expression, recombinant baculovirus.

Stably Express Spider Flagelliform Silk Protein in Bombyx Mori Cell Line by PiggyBac Transposon-Derived Vector

Silkworm BmN4 cells were transfected with the helper plasmid and the piggyBac vector( piggyBac-FLAG) in which was inserted with the spider flagelliform silk expression cassette. Via antibiotic selection, most cells showed stable DsRed-expression. Immuno blot analysis showed that flagelliform silk protein of spider was expressed stably in BmN4 cells. Circular dichroism spectra indicated the existence of β-turn structure in recombinant spider flagelliform protein. The present results suggested that transgenic silkworm directly secreting functional spider silk protein in cocoon by using piggyBac system is feasible.

Identification of the unfolded protein response (UPR)-related genes from Bombyx mori cell lines by a subtractive hybridization approach

The baculovirus expression vector system (BEVS) is one of the powerful insect cell systems for heterologous protein expression. However, over-expression of heterologous proteins in this system sometimes results in protein misfolding and aggregation because of insufficient levels of folding catalysts. In previous study using the differential screening (DS) method, we isolated only 40 differentially expressed genes after treatment with tunicamycin, an unfolded protein response (UPR) inducer. To isolate more protein folding catalysts from insect, we performed suppressive subtractive hybridization (SSH) with untreated and tunicamycin-treated Bm5 cell lines in this study. We could isolate 366 differentially expressed clones by SSH method and produced expressed sequence tags (ESTs). ESTs included the UPR pathway-related genes involved in protein folding, including heat shock proteins, molecular chaperones, foldases, as well as glycosylation and secretory pathway related genes. Identification of the tunicamycin responsive genes using SSH provides more information about the UPR-related genes in insect cells, and will facilitate modifications of the protein folding pathway in the ER to improve heterologous protein expression.

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene ( CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Bombyx moricell line as a model of immune‐system organs

We tested 11 Bombyx mori cell lines for induction of cecropin B gene (CecB) expression. After the immune challenge, CecB expression was induced in seven cell lines. A mixture of the cell-free supernatant from the immune-responsive cell lines and lipopolysaccharide activated a promoter of CecB in the non-immune-responsive cell line, indicating that secreted factor(s) is involved in CecB activation. The expressed sequence tags of one of the immune-responsive cell lines, NISES-BoMo-Cam1, contained genes encoding proteins similar to Relish, Cactus, clip-domain serine protease, serpin, lectin, peptidoglycan recognition protein, 6tox and gloverin, in addition to seven known B. mori immune-inducible genes. These results show that NISES-BoMo-Cam1 cells can be used as an in vitro model of the immune system organs of B. mori.

Use of plant‐derived protein hydrolysates for enhancing growth ofBombyx mori(silkworm) insect cells in suspension culture

The successful suspension culture of the Bombyx mori (silkworm) cell lines Bm5 and BmN4 without FBS (fetal-bovine serum) was first realized in Sf-900 II SFM (serum-free medium) (Gibco BRL, Rockville, MD, U.S.A.) supplemented with a plant-derived protein hydrolysate. The addition of 0.5% HyPep 1510 (Difco Co., Detroit, MI, U.S.A.) and 10 mM glutamine to Sf-900 II SFM at 4 days of culture was found effective in increasing the cell concentration to 8.5x10(6) cells/ml. The replacement of medium with Sf-900 II SFM supplemented with 0.5% HyPep 1510 at 6 days of culture increased the cell concentration by 1.1x10(7) cells/ml. When Sf-900 II SFM was supplemented with 0.5% Hypep 1510, 16 days, which was half of the conventional adaptation time, was sufficient for the B. mori cell line to adapt to SFM and shear stress while maintaining a stable viability. The beta-galactosidase activity in Sf-900 II SFM supplemented with 0.5% Hypep 1510 was 4.9x10(3) units/ml, which was 2-fold higher than that of the FBS-supplemented medium. By SDS/PAGE, only the band corresponding to beta-galactosidase was detected in the sample from the media supplemented with plant-derived protein hydrolysates, while thick bands corresponding to proteins having lower molecular masses than beta-galactosidase were detected in samples from the FBS-supplemented media. These results suggest that plant-derived protein hydrolysates are promising FBS substitutes for enhancing the growth of B. mori cells and facilitating the purification of recombinant proteins produced by baculovirus infection.