Sf9 Cell Line Research Articles

X-tox: An atypical defensin derived family of immune-related proteins specific to Lepidoptera

We report here the isolation in Spodoptera frugiperda ( Lepidoptera) of an immune-related protein (hereafter named Spod-11-tox), characterized by imperfectly conserved tandem repeats of 11 cysteine-stabilized alpha beta motifs (CS- αβ), the structural scaffold characteristic of invertebrate defensins and scorpion toxins. Spod-11-tox orthologs were only found in Lepidopteran species, suggesting that this new protein family (named X-tox) is specific to this insect order. Moreover, phylogenetic analysis suggests that X-tox proteins represent a new class of proteins restricted to Lepidoptera and likely derived from Lepidopteran defensins. In S. frugiperda, analysis of gene expression revealed that spod-11-tox is rapidly induced by infection. However, and conversely to what is known for most insect antimicrobial peptides (AMP), spod-11-tox is mainly expressed in blood cells. Moreover, recombinant Spod-11-tox produced in the Sf9 cell line does not show any antimicrobial activity. Altogether, these results suggest that although X-tox proteins are derived from defensins, they may play a different and still unknown role in Lepidoptera immune response.

Targeting ie‐1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms

RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac-mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection.

Treating Parkinson's the CDNF Way

Lindholm et al . report the cloning, molecular characterization, and tissue expression of a member of a new family of neurotrophins. Through sequence analysis, they identified a gene similar to that encoding mesencephalic-astrocyte-derived neurotrophic factor (MANF, also known as ARMET) and named the new protein conserved dopamine neurotrophic factor (CDNF, which in genomic databases was called ARMETL1 for ARMET-like 1). CDNF was secreted when heterologously expressed in human cultured cell lines or the insect cell line Sf9. Electrospray ionization-mass spectrometry analysis of the secreted protein indicated that the protein formed four disulfide bridges with the eight conserved cysteines. Transcript analysis in mice revealed that CDNF was expressed in several tissues during embryogenesis and postnatally. In the brain, in situ protein analysis showed that the protein was present in the somas of neurons. Although MANF was previously reported to promote survival of dopaminergic neurons in vitro, Lindholm et al . show that in a rat model of Parkinson's disease, injection of CDNF before or after exposure to the neurotoxin prevented or ameliorated symptoms and reduced the loss of dopaminergic neurons in the substantia nigra. P. Lindholm, M. H. Voutilainen, J. Laurén, J. Peränen, V.-M. Leppänen, J.-O. Andressoo, M. Lindahl, S. Janhunen, N. Kalkkinen, T. Timmusk, R. K. Tuominen, M. Saarma, Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo . Nature 448 , 73-77 (2007). [PubMed]

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Functional characterization of human organic anion transporter 4 (hOAT4) in a Baculovirus expression system

In the renal proximal tubule, organic anion transporters (OATs) mediate the removal of endogenous and exogenous toxins from blood for excretion in urine. OATs at the basolateral membrane drive organic anions into the cells, through exchange for dicarboxylates. At the apical membrane, different transporters deliver organic anions to the lumen. hOAT4 resides in the apical membrane, but its role in transport is unclear. Here we describe a new expression system to characterize hOAT4 and other OATs. An insect cell line (Sf9) was infected with Baculovirus containing hOAT4 cDNA under the control of the polyhedrin promoter. Although ABC transporters have been expressed using Baculovirus, this is the first report of insect cell expression of any OAT. Membrane vesicles from infected Sf9 cells transported known hOAT4 substrates (DHEAs, MTX and OTA) in a time-dependent manner. hOAT4-specific accumulation was inhibited by probenecid and increased when vesicles were preloaded with glutarate, suggesting organic anion exchange. In summary, Baculovirus-expressed hOAT4 is functionally consistent with protein from other systems. Isolated membrane vesicles derived from Baculovirus-infected Sf9 cells can therefore be used to characterize the mechanisms by which OATs function. This research was supported by the Intramural Research Program of the NIH, NIEHS.

QSAR and mode of action studies of insecticidal ecdysone agonists†

A series of our SAR and QSAR studies of synthetic moulting hormone agonists, dibenzoylhydrazines (DBH), exhibiting insecticidal/larvicidal activity are reviewed in this article. We prepared a number of analogues where various substituents are introduced into the two benzene rings of DBH and measured their biological activity using various biological systems. Larvicidal activity was against larvae of the rice stem borer Chilo suppressalis and the moulting hormone activity was in terms of the stimulation of N-acetylglucosamine incorporation in a cultured integument system of the same insect species. Binding affinity to the ecdysone receptor was assayed with intact Sf-9 cell lines in which the ADME processes are negligible as well as using receptor proteins obtained by in vitro translation of the responsible cDNA cloned from cell-free preparation of integumentary tissue of C. suppressalis. Variations in the biological activity indices were either correlated between two types of activity or correlated using physicochemical molecular and substituent parameters in terms of the classical QSAR. Comparisons among correlations and with recently revealed X-ray crystallographic findings clearly indicate the physicochemical meaning of parameters significant in the correlation equations to help understanding molecular mechanism of the moulting hormonal action. †Presented at the 12th International Workshop on Quantitative Structure-Activity Relationships in Environmental Toxicology (QSAR2006), 8--12 May 2006, Lyon, France.

Production and purification of functional truncated soluble forms of human recombinant L1 cell adhesion glycoprotein from Spodoptera frugiperda Sf9 cells

L1 is a human cell adhesion glycoprotein involved in the development of the central nervous system that comprises six immunoglobulin-like domains (Ig1–Ig6), five fibronectin-type III (FN1–FN5) domains, a single transmembrane region and a cytoplasmic domain. It contains 20 potential N-glycosylation sites and is heavily glycosylated in a variety of cell types. In this work, seven truncated soluble forms including L1 ectodomain (L1/ECD) and Ig domains 5–6 (L1/Ig5–6) have been constructed by PCR and have been cloned, as well as the full-length form (L1), in the stable expression vector for insect cells pMIB/V5-His-TOPO. Spodoptera frugiperda Sf9 cell lines expressing the truncated forms have been obtained, and all proteins were successfully secreted. L1/ECD and L1/Ig5–6 were produced in shake flasks with productions of 3 and 32 mg/L on the third and fourth day of culture, respectively. When L1/Ig5–6 was produced for four days in 2 L bioreactor 200 mg/L protein were recovered from the supernatants on the fourth day of culture. Affinity-purified L1/ECD and L1/Ig5–6 were immobilized on poly- d-lysine coated coverslips, and were shown to be active in inducing neurite outgrowth from human NT2N neurons. Therefore, correctly folded and functional truncated forms of human L1 have been produced in high amounts from insect cells using a stable expression system.

Function of Spodoptera exigua nucleopolyhedrovirus late gene expression factors in the insect cell line SF-21

We used a well established transient expression assay to test the ability of the baculovirus Spodoptera exigua M nucleopolyhedrovirus (Se MNPV) homologs of Autographa californica MNPV (Ac MNPV) late expression factors ( lefs) to activate a late promoter–reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for Ac MNPV infection but not for Se MNPV. In the assay, 19 Ac MNPV lefs stimulate optimal levels of late gene promoter activity. Se MNPV lef-5 successfully replaced the corresponding Ac MNPV gene in the context of the remaining set of Ac MNPV lefs, whereas Se MNPV dnapol and 39k exhibited partial activity. When all the Se MNPV lefs were assayed together or in the presence of four lefs encoded only in Ac MNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, Se MNPV genomic DNA and the four Ac MNPV-specific lefs stimulated low levels of reporter gene activity. Moreover, Se MNPV IE-1, but not Ac MNPV IE-1, further stimulated late gene expression in the presence of Se MNPV DNA. Ac MNPV IE-1 was able to mediate early gene expression cis-linked to homologous regions ( hrs) derived from Ac MNPV and Se MNPV. In contrast, Se MNPV IE-1 was more specific for Se MNPV-derived hr elements.

Preparation of PLL-PEG-PLL and its application to DNA encapsulation

PLL-PEG-PLL was synthesized by deprotection of the amino group of PLL(Z)-PEG-PLL(Z). The properties of the polyion complex (PIC) micelles comprising of PLL-PEG-PLL and DNA were investigated. The results showed that the core of PIC was formed by PLL and DNA through electrostatic interactions, and PEG was shell of PIC, so PIC micelles in aqueous medium were homogenous with no precipitation even under the neutral condition, and PIC had the characteristic of stability, which were very important in gene therapy. At the same time, the results showed PLL-PEG-PLL could condense DNA, longer PLL blocks were generally more effective at condensation, and the nuclease resistance of DNA was increased remarkably. PIC was spherical with diameter of 10–35 nm, which could be easily taken up by cells. Transfection experiments in vitro showed that the DNA encapsulated could be expressed in the cell line SF9.

SPODOBASE: an EST database for the lepidopteran crop pest Spodoptera.

BackgroundThe Lepidoptera Spodoptera frugiperda is a pest which causes widespread economic damage on a variety of crop plants. It is also well known through its famous Sf9 cell line which is used for numerous heterologous protein productions. Species of the Spodoptera genus are used as model for pesticide resistance and to study virus host interactions. A genomic approach is now a critical step for further new developments in biology and pathology of these insects, and the results of ESTs sequencing efforts need to be structured into databases providing an integrated set of tools and informations.DescriptionThe ESTs from five independent cDNA libraries, prepared from three different S. frugiperda tissues (hemocytes, midgut and fat body) and from the Sf9 cell line, are deposited in the database. These tissues were chosen because of their importance in biological processes such as immune response, development and plant/insect interaction. So far, the SPODOBASE contains 29,325 ESTs, which are cleaned and clustered into non-redundant sets (2294 clusters and 6103 singletons). The SPODOBASE is constructed in such a way that other ESTs from S. frugiperda or other species may be added. User can retrieve information using text searches, pre-formatted queries, query assistant or blast searches. Annotation is provided against NCBI, UNIPROT or Bombyx mori ESTs databases, and with GO-Slim vocabulary.ConclusionThe SPODOBASE database provides integrated access to expressed sequence tags (EST) from the lepidopteran insect Spodoptera frugiperda. It is a publicly available structured database with insect pest sequences which will allow identification of a number of genes and comprehensive cloning of gene families of interest for scientific community. SPODOBASE is available from URL:

Expression and Characterization of Full-length Human Huntingtin, an Elongated HEAT Repeat Protein

Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein. The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix.

Stable RNA Interference in Spodoptera frugiperda Cells by a DNA Vector-based Method

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.

Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21

We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (Ac MNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for Ac MNPV infection. It has been well established that 19 Ac MNPV late expression factors ( lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the Ac MNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an Ac MNPV late promoter–reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding Ac MNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding Ac MNPV homolog in the context of the remaining Ac MNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the Ac MNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.

Two lepidopteran cell lines stably transformed by the ABC transporter gene PDR5 show tolerance to diacetoxyscirpenol

The pleiotropic drug resistance 5 gene (pdr5) encodes a multidrug membrane transporter and plays a very important role in the efflux of a broad range of chemicals in yeast cells. To study the possible function of pdr5 in insect cells, two stably pdr5-transformed lepidopteran insect cell lines, Sf21 and CF-203, were developed. Transcripts of pdr5 were detected in these two lines using Northern blotting and RT-PCR analysis. When cells were treated with the protein synthesis inhibitor diacetoxyscirpenol, the transformed Sf21 and CF-203 cell lines showed increased tolerance to this chemical. However, unlike in yeast cells, ecdysone agonist RH5992 could not be excluded by PDR5, probably because of low expression levels or imperfect incorporation of the recombinant protein in these transformed cell lines.

Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-madiated gene expression in insect cells

The baculovirus expression vector system (BEVS) is used extensively for the production of proteins from exogenous cDNAs. However, BEVS is not ideal for pharmaceutical production of glycoproteins owing to the properties of the N-glycans in the expressed products and that insect cells lack several of the enzymes required for mammalian-type N-glycan synthesis. This study describes the effective mammalian-like production of glycoproteins, such as beta-1,4-galactosyltransferase and alpha-2,6-sialyltransferase, in the insect cell line Sf9.

Cell-cycle-dependent resistance toBacillus thuringiensisCry1C toxin in Sf9 cells

The Sf9 cell line, derived from the moth Spodoptera frugiperda, is highly and specifically sensitive to the Bacillus thuringiensis Cry1C toxin. Upon exposure to Cry1C, ionic pores are formed in the plasma membrane leading to cell swelling and death. Here, we describe a unique transient tolerance to Cry1C of dividing cells, which allowed completion of the division process in the presence of Cry1C. Correlatively, arresting the cells at G2-M phase by nocodazole treatment rendered them insensitive to Cry1C. When the arresting agent was removed, the cells completed their division and gradually regained Cry1C sensitivity. In comparison to normal cells with 1-2% cell-division frequency, the M-phase arrested cells bound less toxin in binding assays. Moreover, no lipid rafts could be isolated from the membranes of M-phase arrested cells. Caveolin-1, identified here for the first time in insect cells, was immunodetected as a lipid raft component of normal cells, but was only present in the membrane-soluble fraction of G2-M-arrested cells. Thus M-phase-linked changes in lipid raft organization may account for diminished Cry1C binding and toxicity. Furthermore, considering the pivotal role of lipid rafts in different cell functions of many cell types, the lack of organized lipid rafts in dividing cells may transiently affect cell susceptibility to pathogens, toxins and other lipid raft-linked functions.

Rational design of an EGF-IL18 fusion protein: Implication for developing tumor therapeutics

Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly 4Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-γ to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics.

Diterpenes from Salvia broussonetii Transformed Roots and Their Insecticidal Activity

The new diterpenes brussonol (1) and iguestol (6alpha,11-dihydroxy-12-methoxy-abieta-8,11,13-triene) (2) with an icetexane and a dehydroabietane skeleton, respectively, have been isolated from hairy root cultures of Salvia broussonetii. Other previously known diterpenes, 7-oxodehydroabietane, 11-hydroxy-12-methoxyabietatriene, taxodione, inuroyleanol, ferruginol, deoxocarnosol 12-methyl ether, cryptojaponol, pisiferal, sugiol, isomanool, 14-deoxycoleon U, 6alpha-hydroxydemethylcryptojaponol, demethylsalvicanol, and demethylcryptojaponol, were also obtained from these roots. The insect antifeedant and toxic effects of several of these compounds were investigated against the insect pests Spodoptera littoralis and Leptinotarsa decemlineata. Additionally, their comparative cytotoxic effects were tested on insect Sf9 and mammalian CHO cells. Demethylsalvicanol (4) was a moderate antifeedant to L. decemlineata, whereas brussonol (1) was inactive. 14-Deoxycoleon U (15) was the strongest antifeedant, whereas demethylcryptojaponol (11) was toxic to this insect. None of these compounds had antifeedant or negative effects on S. littoralis ingestion or weight gains after oral administration. Demethylcryptojaponol (11) was cytotoxic to mammalian CHO and insect Sf9 cell lines, followed by the icetexane derivative brussonol (1), with moderate cytotoxicity in both cases. The remainder of the test compounds showed a strong selective cytotoxicty to insect Sf9 cells, with demethylsalvicanol (4) being the most active.

Grayanane Diterpenoids from the Flowers of Rhododendron molle with Cytotoxic Activity against a Spodoptera frugiperda Cell Line

Two new grayanane diterpenoids, rhodomolins A (1) and B (2), together with two known diterpenoids, rhodomolleins I and rhodojaponin III, were isolated from the flowers of Rhododendron molle. The structures of 1 and 2 were elucidated on the basis of interpretation of spectroscopic data. All compounds were evaluated for cytotoxic activity against the Spodoptera frugiperda cell line Sf-9 and gave IC(50) values in the range 12-80 microg/mL.

Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic™ insect cells

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in Mimic cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation.