Mori Cell Line Research Articles

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.

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuDeltaep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuDeltaep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.

Functional Characterization of a Cactus Homolog from the SilkwormBombyx mori

A cDNA encoding an IkappaB family protein was identified and the full nucleotide sequence was determined in the silkworm Bombyx mori. The IkappaB gene, designated BmCactus, was constitutively expressed mainly in the fat body and hemocytes. Transfection experiments on a B. mori cell line, NIAS-Bm-aff3, with expression vectors containing BmCactus, BmRelA, BmRelB, or the active portion of BmRelish1 showed that activation of the CecB1 gene promoter by either BmRelA or BmRelB, but not the active portion of BmRelish1, was strongly inhibited by BmCactus. In addition, activation of CecB1 gene by autoclaved E. coli in the cultured cells was observed regardless of the presence or absence of BmCactus. A glutathione S-transferase pull-down assay and analysis using a yeast two-hybrid system demonstrated that BmCactus interacted with the BmRel Rel homology domain, but not with the BmRelish Rel homology domain. These results suggest that BmCactus is involved in the Toll signal transduction pathway in B. mori.

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.

Construction of hybrib Autographa californica nuclear polyhedrosis bacmid by modification of p143 helicase

We developed a new hybrid nuclear polyhedrosis virus (NPV) bacmid capable of infecting Spodoptera frugiperda, Tricoplusia ni, and Bombyx mori, and B. mori cell lines for producing hybrid recombinant baculovirus that can carry a gene of interest and express it in a broad range of hosts. A GFP uv-β1,3- N-acetylglucosaminyltransferase 2 fusion gene was expressed successfully in silkworm larvae using this hybrid bacmid. The hybrid NPV bacmid provides an altogether simple and realistically feasible method for large-scale applications using silkworm larvae. It can be easily managed in E. coli, which has no biohazard safety concerns, in addition to the baculovirus-based expression system.

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.

Novel Macula-Like Virus Identified in Bombyx mori Cultured Cells

We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.

A constitutive super-enhancer: homologous region 3 of Bombyx mori nucleopolyhedrovirus

Transient expression assays were carried out to assess the transcriptional enhancement from the immediate-early gene ( ie-1 ) promoters by cis -linked Bombyx mori nucleopolyhedrovirus (BmNPV) homologous region-3 ( hr3 ). The ie-1 promoters were derived from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and BmNPV. Hr3 was placed downstream of the luciferase ( luc ) gene, the resulting plasmids were used to transfect the Spodoptera frugiperda (Sf-21), Bombyx mori (Bm-5, Bm-N) cell lines and 5th instar silkworm larvae mediated by lipofectin. The results revealed that hr3 could stimulate the transcription of homologous BmNPV ie-1 promoter 1970, 2421, 683, and 1059 folds, and the heterologous AcMNPV ie-1 promoter 6462, 4046, 6980, and 605 folds, respectively. These results implied that hr3 was a super enhancer which could function both in vitro and in vivo. The 30-bp imperfect palindrome in hr3 was proved to be the essential structure for enhancing function of hr3 . The utilizing of this promoter–enhancer combination in the baculovirus expression vector system (BEVS) was also carried out to further confirm the function of hr3 enhancer.

A constitutive super-enhancer: homologous region 3 of Bombyx mori nucleopolyhedrovirus

Transient expression assays were carried out to assess the transcriptional enhancement from the immediate-early gene ( ie-1) promoters by cis-linked Bombyx mori nucleopolyhedrovirus (BmNPV) homologous region-3 ( hr3). The ie-1 promoters were derived from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) and BmNPV. Hr3 was placed downstream of the luciferase ( luc) gene, the resulting plasmids were used to transfect the Spodoptera frugiperda (Sf-21), Bombyx mori (Bm-5, Bm-N) cell lines and 5th instar silkworm larvae mediated by lipofectin. The results revealed that hr3 could stimulate the transcription of homologous BmNPV ie-1 promoter 1970, 2421, 683, and 1059 folds, and the heterologous AcMNPV ie-1 promoter 6462, 4046, 6980, and 605 folds, respectively. These results implied that hr3 was a super enhancer which could function both in vitro and in vivo. The 30-bp imperfect palindrome in hr3 was proved to be the essential structure for enhancing function of hr3. The utilizing of this promoter–enhancer combination in the baculovirus expression vector system (BEVS) was also carried out to further confirm the function of hr3 enhancer.

Characterization of a homologue of the Rel/NF-$kappa;B transcription factor from a beetle, Allomyrina dichotoma*1

A cDNA encoding a Rel/NF-κB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reactions (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-κB homologue was designated A. dichotoma (A.d.) Rel A. The amino acid sequence of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% identity with Tribolium castaneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A RHD was confirmed to bind specifically to the NF-κB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. The activity of A.d. Rel A in modulating a gene construct of the A.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing the mutated NF-κB site, suggesting the importance of the interaction between the NF-κB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma.

Characterization of a homologue of the Rel/NF-κB transcription factor from a beetle, Allomyrina dichotoma

A cDNA encoding a Rel/NF-κB homologue was cloned from a beetle, Allomyrina dichotoma, by reverse transcriptase-polymerase chain reactions (RT-PCR) taking advantage of the conserved Rel homology domain (RHD) to synthesize primers. The Rel/NF-κB homologue was designated A. dichotoma ( A.d.) Rel A. The amino acid sequence of the A.d. Rel A RHD was compared with those of insect RHDs. The result showed that it has 70% identity with Tribolium castaneum Dorsal, 66% with Drosophila melanogaster Dorsal, 61% with Anopheles gambiae Gambif1, and 55% with D. melanogaster Dif. A putative phosphorylation site in the RHD, RRPS, and two putative nuclear localization signals were conserved in A.d. Rel A. A recombinant fusion protein containing the A.d. Rel A RHD was confirmed to bind specifically to the NF-κB site of a gene encoding A.d. coleoptericin A, an antibacterial peptide from A. dichotoma. The activity of A.d. Rel A in modulating a gene construct of the A.d. coleoptericin A promoter-luciferase reporter by expressing the A.d. coleoptericin A cDNA in a Bombyx mori cell line was analyzed. The result showed that A.d. Rel A strongly activates the A.d. coleoptericin A gene construct, whereas A.d. Rel A failed to activate the gene construct containing the mutated NF-κB site, suggesting the importance of the interaction between the NF-κB site and A.d. Rel A in the signal transduction for gene expression of antibacterial peptides in A. dichotoma.

A cell-based high-throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds.

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Effect of Virus Dose on Protein Production Profile Using Baculovirus Vector in Bombyx mori Cell Line and Larvae

Effect of Virus Dose on Protein Production Profile Using Baculovirus Vector in Bombyx mori Cell Line and Larvae

Analysis of the 5'-upstream regulatory region of a gene encoding coleoptericin, an antibacterial protein from the beetle, Allomyrina dichotoma(Coleoptera: Scarabaeidae).

A gene encoding coleoptericin, an antibacterial protein from the beetle, Allomyrina dichotoma was cloned and the nucleotide sequence determined. The A. dichotoma (A. d.) coleoptericin genomic clone was full size and contained an intron between a putative transcription initiation site and a signal sequence region. The 5′-upstream regulatory region of the A. d. coleoptericin had a TATA box and other regulatory motifs such as a NF-κB site, a region 1 (R1), a NF-IL6 site, 2 GATA motifs and 3 CATT(A/T) motifs. Expression vectors consisting of different lengths of the regulatory region and a luciferase gene as a reporter were constructed to analyze the induction of gene expression by bacterial lipopolysaccharide (LPS), a strong trigger for insect antibacterial protein genes. A silkworm Bombyx mori cell line was transfected with the expression vectors treated with or without LPS and the luciferase activity measured. Results showed that luciferase activity was dramatically reduced when NF-κB site was deleted. Deletion of R1 resulted in 64% reduction of luciferase activity but no marked effect was observed with GATA motif mutation. These results suggest that the NF-κB site is indispensable for A. d. coleoptericin gene expression and R1 is also necessary for full gene expression.

A Novel Lipopolysaccharide Response Element in the Bombyx mori Cecropin B Promoter

Cecropin B is one of the major antibacterial peptides in the silkworm, Bombyx mori. Transcription of the cecropin B gene (CecB) occurs rapidly after bacterial invasion. Using 235 base pairs (bp) of the CecB promoter region, a kappaB-related protein and two additional DNA-binding complexes (designated F2BPI and F4BP) were identified in nuclear extracts from immunized larval fat body by the electrophoretic mobility shift assay (EMSA) (1). Further EMSA analyses indicated that the F2BPI-binding site was CATTA, and that F2BPI translocated from the cytoplasm to the nucleus after infection. In a recently established B. mori cell line, NISES-BoMo-DZ, 235 bp of CecB promoter linked to a reporter luciferase was activated 6-fold by stimulation with lipopolysaccharide (LPS), which is a major trigger of CecB expression in larvae. Truncation of the F2BPI-binding site from the promoter reduced the activation 2-fold. Deletion of either of two kappaB motifs also reduced promoter activation 2-fold. Elimination of both the F2BPI-binding site and the kappaB motifs resulted in the complete loss of LPS inducibility. These results indicate that the F2BPI-binding site is an LPS-responsive cis-element that is necessary for full activation of CecB.

Novel Bombyx mori cell lines cultivable at 37°C

Four continuous cell lines were established from cultures of embryonic tissue of the silkworm, Bombyx mori, and designated NISES-BoMo-MK, NISES-BoMo-KG, NISES-BoMo-DZ, and NISES-BoMo-OH. The cells were mostly spherical in shape, and could be cultured at 25°C to 37°C. The population doubling time of each cell line was not affected by the temperatures employed, being about one day at both 25°C and 37°C. The karyotype of the cells was typical of lepidopteran cell lines. The number of chromosomes was higher at 37°C than at 25°C. The activity of the enzymes, phosphoglucose isomerase, phosphoglucomutase, isocitrate dehydrogenase, and glucose-6-phosphate dehydrogenase extracted from the cells was higher at 37°C than at 25°C. None of these novel cell lines formed any polyhedra, even with high titer inoculation of Bombyx mori nuclear polyhedrosis virus.

Two key mutations in the host-range specificity domain of the p143 gene of Autographa californica nucleopolyhedrovirus are required to kill Bombyx mori larvae.

Autographa californica nucleopolyhedrovirus (AcMNPV) does not replicate in Bombyx mori cells (Bm5, BmN). We have shown previously that when a short DNA sequence within AcMNPV ORF95, which encodes the viral helicase P143, is replaced with the colinear region of B. mori nucleopolyhedrovirus (BmNPV), AcMNPV gains the ability to replicate in Bm5 cells. To determine the mutational events in the p143 gene required to allow AcMNPV replication in B. mori cells, AcMNPV recombinants produced in Sf9 cells were screened in vivo in B. mori larvae, which are more permissive to baculovirus infection than B. mori cell lines. Eight combinations of mutations were tested and characterization of viral DNA extracted from dead larvae showed that amino acid changes at position 564 and 577 are required to kill B. mori larvae.