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PubMed2000

Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines.

A recombinant AcMNPV containing the green fluorescent protein (gfp) gene under the polyhedrin promoter (polh) was used to investigate the expression of the gfp gene as well as the production of recombinant extracellular virus in 14 continuous insect cell lines, including Heliothis virescens (BCIRL-HV-AM1), Helicoverpa zea (BCIRL-HZ-AM1), Anticarsia gemmatalis (BCIRL-AG-AM1), Trichoplusia ni (TN-CL1), Spodoptera frugiperda (IPLB-SF21), Spodoptera exigua (BCIRL/AMCY-Se-E1 and BCIRL/AMCY-Se-E5), Bombyx mori (BMN), Sf9 (a clone of IPLB-SF21), and five cell line clones of BCIRL-HV-AM1. The susceptibility of the cell lines to the recombinant virus (AcMNPV.GFP) was ascertained by calculating the mean percentage number of green light-emitting cells as well as by TCID50 titration of extracellular virus with fluorescence as a sign of infection. Of the 14 cell lines tested, all were permissive with varying degrees to AcMNPV.GFP, except BCIRL-HV-AMCL2 and BCIRL-HZ-AM1, both grown in serum-containing medium, and BMN, grown in serum-free medium, which were nonpermissive to the virus. Except for BCIRL/AMCY-Se-E1, IPLB-SF21, and four of the five BCIRL-HV-AM1 clones, all the other cell lines (BCIRL-HV-AM1, BCIRL-AG-AM1, TN-CL1, Se-E5, and Sf9) expressed detectable levels of GFP by 48 h postinoculation. The BCIRL/AMCY-Se-E1 and IPLB-SF21 cells, grown in serum-free medium (Ex-Cell 401), expressed detectable levels of GFP at 72 h postinoculation. By contrast, in BCIRL/AMCY-Se-E1 in serum-containing medium (Ex-Cell 401 + 10% FBS [fetal bovine serum]), GFP was detected at 48 h postinoculation. Furthermore, TN-CL1 cells produced the largest mean percentage number of fluorescent (76.6%) cells in both serum-containing and serum-free medium (64.8%) at 120 h postinoculation. All the BCIRL-HV-AM1 clones showed no GFP expression until 96 h postinoculation, and only then about 1% of the cell population fluoresced. The mean extracellular virus (ECV) production at 120 h postinoculation was highest in BCIRL/AMCY-Se-E5 cells grown in Ex-Cell 401 + 10% FBS (37.8 x 10% TCID50/ml) followed by BCIRL-HV-AM1 in TC199-MK (33.4 x 10(6) TCID50/ml). Only the BCIRL-HV-AMCL3 clone produced any substantial level of ECV at 120 h postinoculation (16.9 x 10(6) TCID50/ml). However, there was no significant correlation between ECV production and the mean percentage number of fluorescent cells. This study provides further information on the susceptibility of 14 insect cell lines to a recombinant AcMNPV containing the green fluorescent protein gene. This information might avail researchers with information to facilitate decisions as to what other cell lines are available for in vitro studies of the gfp gene.

Animals↗

PubMed1994

Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin.

During postembryonic development of insects, molting cycles affect epidermal cells with alternate periods of proliferation and differentiation. Cells of the cell line established from imaginal discs of the Indian meal moth (IAL-PID2) differentiate under the action of the molting hormone, 20-hydroxyecdysone, in a manner that is meaningful in terms of the development of the tissue from which they were derived. In particular, the hormone caused an accumulation of the cells in the G2 phase of their cycle and induced the formation of epithelial-like aggregates and the synthesis of specific proteoglycans. Recent discovery of members of the insulin superfamily in insects and the role of growth factors played by this family of molecules in vertebrates led us to check for their potential effects on IAL-PID2 cell cycle regulation. On the one hand, our results showed that insulin was involved in partial resumption of the cell cycle after an arrest caused by serum deprivation, but that other growth factors present in fetal calf serum were needed for full completion of mitosis. On the other hand, the cytostatic effect of 20-hydroxyecdysone was reversible, and, prior exposure of the cells to the hormone allowed the cells to complete one cell cycle in serum-free medium. These results suggest that the production of autocrine growth factors induced by ecdysteroids could circumvent the absence of serum. This cell culture model provides potential for further study of interactions between ecdysteroids and growth factor homologs during differentiation of insect epidermal cells.

Animals↗

PubMed2001

An anti-Bcl-2 antibody prevents 2-deoxy-D-ribose-induced apoptosis in the IPLB-LdFB insect cell line.

Confocal microscopy reveals that the anti-Bcl-2 antibody (pAb) is able to diffuse across the plasma membrane of the fat body cell line IPLB-LdFB from the insect Lymantria dispar, demonstrating the presence of Bcl-2-like molecules in the cytoplasm. Immunoperoxidase procedure confirms the cellular localization. Furthermore, an immunoprecipitation corresponding to a molecular weight of 29 KDa is observed with western blot analysis using the anti-Bcl-2 pAb. Cytofluorimetric experiments show that anti-Bcl-2 pAb counteracts 2-deoxy-D-ribose-induced apoptosis and provokes morphological changes in the insect cell line, i. e. a reduction in cell size, the disappearance of the vacuola and changes in shape. At the same time, the antibody provokes mitochondrial membrane depolarization, and N-acetyl-L-cysteine is unable to reconstitute the physiological conditions. The present findings suggest that Bcl-2-like proteins play a main role in maintaining of the integrity of cellular components, e.g. mitochondria, rather than in controlling programmed cell death.

Animals↗

PubMed1999

Construction of modular and versatile plasmid vectors for the high-level expression of single or multiple genes in insects and insect cell lines.

We have constructed a series of plasmid vectors for the expression of foreign genes in insects or insect cell lines. We incorporated the Drosophila hsp70 and actin 5C promoters, as well as the hr5 enhancer-driven baculovirus ie1 promoter, into plasmids that allow convenient cloning of heterologous genes into multiple cloning sites. We combined these promoters with either a short, double poly-adenylation site derived from the Heliothis virescens p63 chaperonin gene, or with a fusion of the small t intron with the early 3' untranslated region and poly-adenylation sites of SV40. Unique eight base cutter restriction sites flanking the promoters and poly-adenylation sequences make it possible to transfer the entire transcription units into other sequence contexts, for example, into transposable elements or into other plasmids bearing selectable marker genes. It is also convenient to combine two of our transcription units on the same plasmid in order to express multiple genes simultaneously. To test the ability of our vectors to drive expression of reporter genes, luciferase derivatives were made of the expression plasmids and introduced into Aedes albopictus C6/36 cells by electroporation or into Anopheles gambiae embryos by biolistic particle bombardment. All three promoters directed high levels of luciferase expression. However, there were differences in their relative activities in the two experimental systems. In C6/36 cells, the actin 5C and hr5-ie1 promoters were significantly more active than the hsp70 promoter. In Anopheles embryos, hsp70 and actin 5C had maximal activities, while hr5-ie1 was weaker. We also found that the constructs containing the SV40 small t intron and early 3' untranslated region sequences had higher expression levels than their counterparts containing the Heliothis poly-adenylation sequence. Our most active construct combines the actin 5C promoter with the SV40 intron and 3' untranslated region sequences. This vector was also used to drive expression of a visible marker, the enhanced green fluorescent protein gene, resulting in readily visible green fluorescent protein expression in C6/36 cells.

3' Untranslated Regions↗

PubMedDate not supplied

An insect cell line discrimination method by RAPD-PCR.

RAPD-PCR with a tenmar single primer for discrimination of insect cell lines was devised. The base sequence of the primers used were TTCGAGCCAG, CGGCATCTAC, GAACGGACTC, and TGAGTGGGTG (GC contents were 60%). Genome DNA was extracted by modified Landry et al. (1993) method. The reaction mixture consisted of 10 microliters buffer, 8 microliters dNTP mixture (2.5 mM each), 4 microliters primer (50 microM), Taq DNA polymerase (2.5 units), 1 microliter template DNA; and the reaction was run at 94 degrees C for 2 min (denaturation), followed by 31 cycles of 94 degrees C for 1 min, 42 degrees C for 1 min (annealing), and 72 degrees C for 2 min (extension) and terminated with 72 degrees C for 7 min. By developing the reaction products with agarose gel electrophoresis, it became evident that DNA fragments were amplified with all the primers used. Among four primers, the second primer was selected as a suitable primer for distinguishing cell lines. With this method, cell lines derived from different species were clearly distinguished.

Animals↗

PubMed2006

Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high-throughput screening platforms for drug and insecticide discovery.

Insect cell-based expression systems are prominent amongst current expression platforms for their ability to express virtually all types of heterologous recombinant proteins. Stably transformed insect cell lines represent an attractive alternative to the baculovirus expression system, particularly for the production of secreted and membrane-anchored proteins. For this reason, transformed insect cell systems are receiving increased attention from the research community and the biotechnology industry. In this article, we review recent developments in the field of insect cell-based expression from two main perspectives, the production of secreted and membrane-anchored proteins and the establishment of novel methodological tools for the identification of bioactive compounds that can be used as research reagents and leads for new pharmaceuticals and insecticides.

Animals↗

PubMed2003

Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines.

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.

Alkaline Phosphatase↗

PubMed2003

Functionality of the 5'- and intergenic IRES elements of cricket paralysis virus in a range of insect cell lines, and its relationship with viral activities.

Cricket paralysis virus (Dicistroviridae: Cripavirus) (CrPV) naturally has a wide range of insect hosts which is reflected in its ability to infect several cultured insect cell lines. The expression of viral gene products is controlled by two kinds of internal ribosome entry site (IRES) elements, 5' and intergenic (IG). Using seven cultured cell lines we tested the functionality of both IRES elements by transfection with bi-cistronic RNA constructs. In six of the seven cell lines, expression initiated from both IRES's was significantly higher than that from a control construct and in five of these six lines the expression from the 5'-IRES was higher than that from the IG-IRES. Permissiveness of each of the cell lines for replication of CrPV was tested by infection with purified virions and transfection with viral RNA. Only three of the cell lines were fully permissive for CrPV replication and no correlation between permissiveness and IRES activity was apparent. These results suggest that while IRES function is required for permissiveness, additional cellular and/or viral factors, involved in processing of viral products, packaging of viral particles and interacting with the cap-dependent translation machinery of host cells, are necessary for CrPV to be able to replicate in any given cell.

5' Untranslated Regions↗

PubMed1996

Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species.

Previous studies (Mertens et al., Virology 157, 375-386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately x 10(-4)) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species of Culicoides that can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately x 10(3)). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10 drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennis and Culicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertens et al., Virology 157, 375-386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately x 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived from Aedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed.

Animals↗

PubMed2002

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB.

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by Western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c.

Androstadienes↗

PubMed2003

Interaction of insect trypanosomatids with mosquitoes, sand fly and the respective insect cell lines.

Interaction experiments between hematophagous insects and monoxenous trypanosomatids have become relevant, once cases of human infection involving these protozoa have been reported. Moreover, investigations related to the interaction of insects with trypanosomatids that harbour an endosymbiotic bacterium and thereby lack the paraflagellar rod structure are important to elucidate the role of this structure in the adhesion process. In this work, we compared the interaction of endosymbiont-bearing trypanosomatids and their aposymbiotic counterpart strains (without endosymbionts) with cell lines of Anopheles gambiae, Aedes albopictus and Lutzomyia longipalpis and with explanted guts of the respective insects. Endosymbiont-bearing strains interacted better with insect cells and guts when compared with aposymbiotic strains. In vitro binding assays revealed that the trypanosomatids interacted with the gut epithelial cells via flagellum and cell body. Flagella attached to the insect gut were enlarged, containing electrondense filaments between the axoneme and flagellar membrane at the point of adhesion. Interactions involving the flagellum lacking paraflagellar rod structure were mainly observed close to tight junctions, between epithelial cells. Endosymbiont-bearing trypanosomatids were able to colonise Aedes aegypti guts after protozoa feeding.

Aedes↗

PubMed2005

Application of a radial-flow bioreactor in the production of beta1,3-N-acetylglucosaminyltransferase-2 fused with GFPuv using stably transformed insect cell lines.

An RFB (radial-flow bioreactor) with a reactor volume of 5 ml was applied to produce human beta3GnT (beta1,3-N-acetylglucosaminyltransferase) using two stably transformed insect cell lines. When air was supplied to the RFB, cell growth stopped at 4 days of culture and beta3GnT was not detected. However, with a supply of pure oxygen, the cell concentration, assumed from glucose consumption, increased by 1.3x10(7) cells/ml. Insect cells attached to poly(vinyl alcohol) matrixes packed in the RFB and grew confluently; 5.6 m-units/ml beta3GnT was produced under the conditions of pure oxygen supply and addition of glucose and glutamine. This RFB was first applied in beta3GnT production using stably transformed insect cells. The amount of beta3GnT production in only a 5 ml-scale RFB was comparable with that of a 100 ml shaking flask culture.

Animals↗

PubMed2005

Cell-death mechanisms in the IPLB-LdFB insect cell line: a nuclear located Bcl-2-like molecule as a possible controller of 2-deoxy-D-ribose-mediated DNA fragmentation.

In the IPLB-LdFB insect cell line, oncosis and apoptosis are the two pre-mortal processes, whereas necrosis is the post-mortem condition. As found in mammals, adenosine triphosphate depletion of insect cells by oligomycin A induces oncosis. The apoptotic inducer 2-deoxy-D: -ribose (dRib) provokes cell death through an intrinsic apoptotic pathway similar to that observed in mammalian models and results in oligonucleosomal DNA fragmentation. The addition to insect cells of an anti-Bcl-2 polyclonal antibody known to prevent dRib-mediated apoptosis abolishes DNA fragmentation, whereas cytochrome c release and the increase in a caspase 3-like activity are still detectable. These and previous findings suggest a double role for the Bcl-2-like molecule in IPLB-LdFB, i.e. the maintenance of mitochondrial integrity and the control of apoptotic machinery at the nuclear level.

Animals↗

PubMed2001

Evidence of oxidative stress following the viral infection of two lepidopteran insect cell lines.

The infection of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) resulted in increased levels of lipid hydroperoxides and protein carbonyls. In addition, the viral infection resulted in a significant decrease in the reduced glutathione to oxidized glutathione (2GSH/GSSG) ratio. These results are all consistent with an increased level of oxidative stress as a result of the viral infection. It was also observed that the oxidative damage corresponded to reduced cell viability, i.e., the results are consistent with the premise that oxidative damage contributes to cell death. Finally, the measured intracellular activities of most of the antioxidant enzymes, specifically manganese superoxide dismutase (MnSOD), ascorbate peroxidase (APOX), and catalase (CAT, not present in Sf-9 cells), did not significantly decrease following viral infection. In contrast, the measured activity of copper-zinc superoxide dismutase (CuZnSOD) decreased in the Sf-9 and Tn-5B1-4 cells following AcMNPV infection.

Animals↗

PubMed1978

Cell cycle analysis and synchronization of the TN-368 insect cell line.

A cell cycle analysis of the Trichoplusia ni (TN-368) insect cell line is described. By means of autoradiography and percent labeled metaphase data, the cell cycle parameters were determined to be as follows: S, 4.5 hr; G2, 8.5 hr; M, 0.5 hr; G1, 1.0 hr; the total cell time being 14.5 hr. A synchronization procedure using 50 mM thymidine in a double block procedure was used to provide a method of obtaining a large number of cells in particular cell cycle phases, especially S and G2.

Animals↗

PubMed1993

Comparative recombinant protein production of eight insect cell lines.

A recombinant Autographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae and Estigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, the T. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than the S. frugiperda-derived Sf9 or Sf2l cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.

Alkaline Phosphatase↗

PubMed1999

Semliki Forest virus as an expression vector in insect cell lines.

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

Aedes↗

PubMed1991

Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9.

The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [3H]glucosamine and infected with a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal beta 1-3GalNAc constituting 17 and 66% of the total number of structures, respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked alpha 1-6 to the asparagine-linked N-acetylglucosamine.

Animals↗