Sf9 Cell Line Research Articles

RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles.

Conformationally complex membrane proteins (MPs) are therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of virus-like particles (VLPs), preserving their native lipidic environment. Here, we implemented a site-specific recombinase-mediated cassette exchange (RMCE) strategy to establish a reusable HIV-1 Gag-expressing insect cell line for fast production of target MPs on the surface of Gag-VLPs. The Sf9 cell line was initially tagged with a Gag-GFP-expressing cassette incorporating two flipase recognition target sites (FRTs), one within the fusion linker of Gag-GFP. The GFP cassette was afterwards replaced by a Cherry cassette via flipase (Flp) recombination. The fusion of Gag to fluorescent proteins enabled high-throughput screening of cells with higher Gag expression and Flp-mediated cassette exchange ability, while keeping the functionality of the VLP scaffold unaltered. The best cell clone was then Flp-recombinated to produce Gag-VLPs decorated with a human β2-adrenergic receptor (β2AR). Release of a fluorescently labeled β2AR into the culture supernatant was confirmed by immunoblotting, and its co-localization with Gag-VLPs was visualized by confocal microscopy. Furthermore, the differential avidity of β2AR-dsplaying Gag-VLPs versus "naked" Gag-VLPs to an anti-β2AR antibody measured by ELISA corroborated the presence of β2AR at the surface of the Gag-VLPs. In conclusion, this novel insect cell line represents a valuable platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.

Cell lines from diamondback moth exhibiting differential susceptibility to baculovirus infection and expressing midgut genes.

Six new cell lines were established from embryonic tissues of the diamondback moth, Plutella xylostella (L.). The cell lines showed differential characteristics, including growth in attachment or in suspension, susceptibility to a baculovirus infection and expression of genes involved in the glucosinolate detoxification pathway in P. xylostella larvae. Five of the cell lines grew attached to the culture flask and one cell line grew unattached as a suspension cell line. The cell lines had population doubling times ranging from 18 to 23 h. Among five of the P. xylostella cell lines examined for infection of a nucleopolyhedrovirus from Autographa californica, AcMNPV, four cell lines were highly susceptible to AcMNPV infection, but one was only semi-permissive to AcMNPV infection. The production of two recombinant proteins, a β-galactosidase of bacterial origin and a secreted alkaline phosphatase of eukaryotic origin, in the P. xylostella cell lines was examined in comparison with that in the cell line Sf9 which is commonly used for recombinant protein production. In the P. xylostella cell lines, expression of three important midgut genes involved in the glucosinolate detoxification pathway, including the glucosinolate sulfatase genes GSS1 and GSS2 and the sulfatase modifying factor gene SUMF1, was detected. The P. xylostella cell lines developed in this study could be useful in in vitro research systems for studying insec-virus interactions and complex molecular mechanisms in glucosinolate detoxification and insect-plant interactions.

Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin

As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

Developing global regression models for metabolite concentration prediction regardless of cell line.

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc.

Resistance irrelevant CYP417A2v2 was found degrading insecticide in Laodelphax striatellus.

Cytochrome P450 monooxygenases (CYPs) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYPs which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYPs viz. CYP417A2v2, CYP425A1v2, and CYP4DJ1 from CYP4 family of Laodelphax striatellus were randomly selected for experiments. CYP417A2v2 and CYP425A1v2 were found expressed successfully in Sf9 cell line while CYP4DJ1 was not expressed successfully and out of two expressed CYPs, only CYP417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, p‐nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min−1 mg protein−1, respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP417A2v2. Incubation of imidacloprid with CYP417A2v2 of L. striatellus and subsequent HPLC, LC‐MS, and MS/MS analysis revealed the formation of imidacloprid metabolites, that is, 4′ or 5′hydroxy‐imidacloprid by hydroxylation. This result implies the exemption of CYPs character that it is not always, all the CYPs degrading insecticides being selected and overexpressed in resistant strains and the degrading CYPs without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYPs in insecticide degradation can provide insight for better understand of insecticide resistance development.

Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line.

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

Selection and validation of reference genes for RT-qPCR analysis in Sf9 cell line infected by Heliothis virescens ascovirus 3h (HvAV-3h)

The real-time quantitative reverse transcription PCR (qRT-PCR) is an efficient and pervasive technique for analysis gene expression. Despite the fact that this technique has been extensively used to explore the gene functions, the stability of the reference genes still requires being validated at different experimental conditions. This research aims to confirm that the expression stability of seven candidates reference genes under different concentrations sterile hemolymph containing virus (2, 4, 6, 108-fold) infects the Spodoptera frugiperda cells line (Sf9). The appropriate reference genes as endogenous controls were determined through four computational algorithms (ΔCt, NormFinder, BestKeeper, and geNorm) and an integrated online software RefFinder. According to results of this study, TUB and ACT1 were proved to be the most stable reference genes in E2 fold and E4 fold respectively. PPI was the best reference gene in E6 and E8 fold, 28S and TUB were the most stable pair reference genes through all samples. These results can facilitate basic researches of the molecular mechanism of HvAV-3h and also prove to be helpful for the standard RT-PCR method.

Substrate and method dependent inhibition of three ABC-transporters (MDR1, BCRP, and MRP2)

Drug transport and drug-drug interactions (DDI) with human ABC transporters are generally investigated in mammalian cell lines or inverted membrane vesicles from insect cells (Sf9) overexpressing the transporter of interest. In this study, we instead used membrane vesicles from human embryonic kidney cells (HEK293) overexpressing wild type MDR1/Pgp (ABCB1), BCRP (ABCG2), and MRP2 (ABCC2) with the aim to study the concentration dependent inhibition of shared and prototypic probe substrates. We first investigated 15 substrates and identified estrone-17-beta-glucorinide (E17G) as shared substrate. Nine specific and general inhibitors were then studied using E17G and prototypic probe substrates. The results were compared with those previously obtained in Sf9 vesicles and cell lines of canine (MDCKII) and human (Saos-2) origin. For the majority of inhibitors, Ki values differed <10-fold between E17G and probe substrates. Significant differences in Ki values were observed for about one third of the inhibitors. The transport inhibition potencies in HEK293 vesicles were in good agreement with those obtained in Sf9 vesicles. Large differences were found in the inhibition potencies observed in the vesicular systems compared to the cellular systems. Nevertheless, the rank order correlations between the different experimental systems were generally good. Our study provides further information on substrate dependent inhibition of ABC-transporters, and suggests that simple ranking of compounds can be used as a tier one approach to bridge results obtained in different experimental systems.

Toxicity of the mycotoxin fumonisin B1 on the insect Sf9 cell line

Fumonisins are a type of mycotoxin produced by Fusarium spp., mainly F. proliferatum and F. vertieilliodes, and represent a potential hazard to the health of animals and human beings. The toxicity and mechanism of action of fumonisins is ambiguous, and it is unclear whether fumonisins are toxic to insect cells. This study examines the toxicity of fumonisin B1 (FB1) and its mechanism of action in the Spodoptera frugiperda Sf9 cell line. We found that FB1 inhibited Sf9 cellular proliferation and arrested cell growth at the G2/M phase. Morphological observation showed that FB1 induced swelling, vacuole formation, and loss of adhesion in Sf9 cells. Flow cytometry analysis showed that FB1 caused depolarization of the cell membrane potential and hyperpolarization of the mitochondrial membrane potential. To uncover potential genes associated with the molecular mechanisms of FB1, 41 differentially expressed genes were identified by transcriptome analyses after FB1 treatment. These genes are putatively involved in detoxification metabolism, insect hormone regulation, cell apoptosis, and other related processes. Finally, six differentially expressed genes were chosen and validated by quantitative real-time PCR (QRT-PCR). Our test could provide a reference for other kinds of insect cells studies on FB1 stress. At the same time, our studies try to provide a possible for FB1 as a precursor compounds of biological insecticide.

Identification of newly established Spodoptera littoralis cell lines by two DNA barcoding markers.

Cell line authentication is crucial in determining the identity of cell lines and detecting any cross-contamination. The identity of three newly established Spodoptera littoralis cell lines (Spli-C, Spli-B, and Spli-S) was confirmed by DNA fingerprinting. In this study, we used two universal primers sets to amplify two DNA fragments in different positions in the mitochondrial cytochrome C oxidase 1 gene (COI). The PCR reaction succeeded in amplifying two target DNA amplicons. The first amplicon had ~650bp, while the second had ~410bp. By comparing the obtained informative sequences with those in the GenBank sequence database, the results showed 100% similarity between the S. littoralis cell lines and their host. The same similarity ratio was observed between the Sf21, Tni, and Cp cell lines, which are used widely, and their hosts. The informative sequences were then used for phylogenetic analyses. In addition to the high efficiency of this technique, it showed high reproducibility in two different laboratories. DNA barcoding using the two sets of the universal primers used in this study can be a fast and a reliable method for insect cell line identification.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche.

Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca2+ release effect in Sf9 cells.

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro, in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca2+-Mg2+-ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms.

Transcriptome Analysis of the Intracellular Facultative Pathogen Piscirickettsia salmonis: Expression of Putative Groups of Genes Associated with Virulence and Iron Metabolism.

The intracellular facultative bacteria Piscirickettsia salmonis is one of the most important pathogens of the Chilean aquaculture. However, there is a lack of information regarding the whole genomic transcriptional response according to different extracellular environments. We used next generation sequencing (NGS) of RNA (RNA-seq) to study the whole transcriptome of an isolate of P. salmonis (FAVET-INBIOGEN) using a cell line culture and a modified cell-free liquid medium, with or without iron supplementation. This was done in order to obtain information about the factors there are involved in virulence and iron acquisition. First, the isolate was grown in the Sf21 cell line; then, the bacteria were cultured into a cell-free liquid medium supplemented or not with iron. We identified in the transcriptome, genes associated with type IV secretion systems, genes related to flagellar structure assembly, several proteases and sigma factors, and genes related to the development of drug resistance. Additionally, we identified for the first time several iron-metabolism associated genes including at least two iron uptake pathways (ferrous iron and ferric iron uptake) that are actually expressed in the different conditions analyzed. We further describe putative genes that are related with the use and storage of iron in the bacteria, which have not been previously described. Several sets of genes related to virulence were expressed in both the cell line and cell-free culture media (for example those related to flagellar structure; such as basal body, MS-ring, C-ring, proximal and distal rod, and filament), which may play roles in other basic processes rather than been restricted to virulence.

Biogenic silver nanoparticles mediated stress on developmental period and gut physiology of major lepidopteran pest Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)—An eco-friendly approach of insect pest control

In the present work, laboratory trial was carried out to determine the cytotoxicity of nanoparticles on Spodoptera frugiperda-21cell lines (SF-21) by MTT assay, developmental influence and gut physiology like nutritional index parameters approximate digestibility, efficiency of conversion ingested and digested food, gut enzymes, gut microbiota against major lepidopteran polyphagous pest Spodoptera litura (Fab.) (Lepidoptera: Noctuidae). Silver nanoparticles synthesized from Pomegranate (Punica granatum) peel extract and the various analytical techniques used to characterize the synthesized nanoparticles revealed the uniform, monodispersive, highly stable particles with the size range of 14–28nm. Nanoparticles at different concentration tested on SF-21cell lines and life stages of Spodoptera litura revealed that the nanoparticles were highly effective against all the tested parameters as dose dependent manner by showing effective inhibition of cell viability on SF-21 cell line, larval and pupal mortality, least lethal concentration 50 (LC50), lethal time 50 (LT50), reduced larval, pupal period, reduced adult emergence and adult longevity. Gut physiology study on third instar of Spodoptera litura showed distinct reduction effect on all the tested parameters.

English

The purpose of this research was to investigate the effect of crude extracts from the root of Stemona tuberosa Lour. on the replication of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Cytotoxicity of crude hexane, dichloromethane and ethanol extracts from the root of S. tuberosa was tested against Spodoptera frugiperda cell line (Sf9) using MTT assay. The cytotoxic effect, represented as CC50 (µg/ml) was observed after 48 and 96 h. It was shown that dichloromethane extract was more toxic than hexane and ethanol extracts and 96-h CC50 for the dichloromethane extract was 1,708.98 µg/ml. Crude dichloromethane extract at the concentration of 31.25 µg/ml was then tested on AcMNPV. The extract was added after 1 h post-infection of AcMNPV at the multiplicity of infection (MOI) of 2, in Sf9 cell line cultivated in vitro. No significant difference between the percentage of infected cells in the control and the test sample with crude dichloromethane extract was found. The average number of polyhedra (OBs/ml) in the control (5.11×106±0.63 OBs/ml) was not significantly different from the test sample (4.19×106±0.31 OBs/ml). However, there was significant difference between the average virus titer (budded virus, BV) in the control (2.06×108±0.71 PFU/ml) and the test samples (2.65×108±0.79 PFU/ml). Crude dichloromethane extract (31.25 µg/ml) did not toxic to Sf9 cells but it could enhance AcMNPV replication in Sf9 cell line cultivated in vitro. It could be concluded that S. tuberosa can be a good candidate for developing the insect virus production in vitro for controlling insect pests. Key words: AcMNPV, crude extract, cytotoxicity test, by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.

Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens

Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.

Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors.

The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

549. Fine Tuning of Transduction Efficiency of rAAV Vectors via Modulation of Capsid Composition

Recombinant Adeno-associated virus (rAAV) vectors have emerged as one of the most versatile and successful gene therapy delivery vehicles. Even though the industry is poised for the expansion into several application areas represented by orphan diseases, a simple and scalable rAAV production technology is still lacking. We have recently developed the OneBac system to allow scalable, high-titer production of the full range of rAAV serotypes by infection of stable insect Sf9 cell lines with a single baculovirus. Some of the serotypes produced in this system, however, were characterized by a low transduction efficiencies compared to HEK 293-derived vectors. Here we describe an approach for resolving this drawback by modulating the ratios of VP1:VP2:VP3 capsid composition to derive particles with a higher VP1 content. This is accomplished by modifying a canonical Kozak sequence preceding VP1 ATG start codon. A range of Kozak sequences has been tested and the most favorable have been identified for AAV5 and AAV9 serotypes. These newly designed capsid genes were incorporated into Sf9 stable cell lines mediating packaging of AAV5 and AAV9 vectors which exceeded transduction efficiencies of HEK 293-derived counterparts by 2-3 folds. Curiously, the optimal ratios of VP1:VP2:VP3 were serotype-specific requiring fine-tuning to achieve a compromise between higher VP1 content mediating higher transduction potencies vs. lower packaging efficiencies impeding production yield. In summary, we have developed a novel approach of significantly enhancing biological potencies of AAV vectors derived from baculovirus system thus facilitating translational applications.