Identification of insect cell lines and cell‐line cross‐contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR‐RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR‐based method used three species‐specific primer sets, Ly‐ITS1/Ly‐ITS2, ITS1‐1/Ld‐ITS1 and Sf9‐F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell‐line cross‐contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell‐line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.     

In vitro rearing of Edovum puttleri,an egg parasitoid of the Colorado potato beetle,on artificial diets: Effects of insect cell line‐conditioned medium

Effects of conditioned media prepared from cell lines derived from 11 insect species (six families, three orders) on the in vitro growth and development of the egg parasitoid Edovum puttleri were investigated. The parasitoid exhibited significantly different responses to the various insect cell line‐conditioned media that were incorporated into the artificial diets. When cell lines were derived from embryos, higher percentages of 3rd instars and prepupae were observed than when cell lines were derived from fat body or ovaries. Medium conditioned with cell line IPLB‐CPB2 derived from the embryos of the Colorado potato beetle produced the best result. Preconditioning time was important. In general, 5 days of preconditioning appeared to be optimal. The growth‐ and development‐promoting effect may have resulted from growth factors or growth‐supporting factors produced/ released by the insect cell lines into the culture medium. Upon storage at 0–4°C for 7–14 days, the ability of cell line‐conditioned medium to promote development beyond the second instar was greatly reduced (approximately 10–55%). Our studies demonstrated that to support the in vitro growth and development of E. puttleri, insect hemolymph could be successfully replaced with insect cell line‐conditioned medium. These findings should facilitate the development of a cost‐effective mass‐rearing system for E. puttleri and/or other parasitoids. Arch. Insect Biochem. Physiol. 40:173–182, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.     

Production of ethanol by cultured insect cells

Summary Proton Nuclear Magnetic Resonance (¹H-NMR) Analysis of insect cell culture media used for cultivating insect cell lines derived from the fleshflySarcophaga peregrina, swallowtail butterflyPapilio xuthus, and cabbage armywormMamestra brassicae revealed that ethanol appeared in the medium as the cultures aged. By incorporating [¹³C-1]-glucose into the media, we pursued¹³C-NMR spectrograms to show that the ethanol was derived from glucose. Thus, it became evident that the insect cells culturedin vitro produce ethanol from glucose as a metabolite.     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

Comparative genomics of mutualistic viruses of Glyptapanteles parasitic wasps

Background

Polydnaviruses, double-stranded DNA viruses with segmented genomes, have evolved as obligate endosymbionts of parasitoid wasps. Virus particles are replication deficient and produced by female wasps from proviral sequences integrated into the wasp genome. These particles are co-injected with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and, thereby, survival of proviral DNA. Here we characterize and compare the encapsidated viral genome sequences of bracoviruses in the family Polydnaviridae associated with Glyptapanteles gypsy moth parasitoids, along with near complete proviral sequences from which both viral genomes are derived.

Results

The encapsidated Glyptapanteles indiensis and Glyptapanteles flavicoxis bracoviral genomes, each composed of 29 different size segments, total approximately 517 and 594 kbp, respectively. They are generated from a minimum of seven distinct loci in the wasp genome. Annotation of these sequences revealed numerous novel features for polydnaviruses, including insect-like sugar transporter genes and transposable elements. Evolutionary analyses suggest that positive selection is widespread among bracoviral genes.

Conclusions

The structure and organization of G. indiensis and G. flavicoxis bracovirus proviral segments as multiple loci containing one to many viral segments, flanked and separated by wasp gene-encoding DNA, is confirmed. Rapid evolution of bracovirus genes supports the hypothesis of bracovirus genes in an 'arms race' between bracovirus and caterpillar. Phylogenetic analyses of the bracoviral genes encoding sugar transporters provides the first robust evidence of a wasp origin for some polydnavirus genes. We hypothesize transposable elements, such as those described here, could facilitate transfer of genes between proviral segments and host DNA.     

Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

Summary Our aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10⁻¹¹–10⁻³ M and with acetic acid (10⁻⁵–10⁻¹ M), acetaldehyde (10⁻¹⁰–10⁻⁴ M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Development and characterization of a continuous cell line,AFKM-On-H,from hemocytes of the European corn borer <Emphasis Type="BoldItalic">Ostrinia nubilalis</Emphasis> (Hübner) (Lepidoptera,Pyralidae)

The corn borer, Ostrinia nubilalis, is a very important pest in different countries, and the in vitro system of the insect could be a useful tool for isolation and characterization of the pathogens and physiological responses of the insect. In this context, a cell line was derived from the hemocytes of the European corn borer and was named AFKM-On-H for, respectively, O. nubilalis, Armand Frappier, King Mongkut Institutes, and Hemocytes. This cell line was initiated and maintained in Ex-Cell 400 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells, mostly spherical in shape, not firmly attached to the plastic culture flasks, were passaged up to 200 times by repeated gentle pipetting of the cells. The doubling times at the 80th and 125th passages at 28°C and at the 122th and 169th passages at 25°C were 40, 29, 35, and 34 h, respectively. The AFKM-On-H cell line was further characterized by the morphology, karyotype, random amplified polymorphic DNA analysis, and isozyme profiles. Susceptibility of the cell line to cytoplasmic polyhedrosis viruses (CPV) Euxoa scandens (EsCPV), Dendrolimus punctatus (DpCPV), and Choristoneura fumiferana (CfCPV); nuclear polyhedrosis viruses [Autographa californica (AcMNPV) wild type and recombinant, Antherea yammamai (AnyaNPV)]; and Chilo iridescent virus was demonstrated. Relative sensitivities of the cell line to Bacillus thuringiensis and Metarhizium anisopliae toxins and effects of the molting hormone 20-hydroxyecdysone on this new hemocyte cell line were characterized.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.     

Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.     

喙尾琵琶甲肠道来源链霉菌(Streptomyces sp.)BPA71次级代谢产物的分离鉴定及其生物活性评价

【背景】昆虫是世界上种类最多、肠道菌群资源最丰富且多样的动物类群之一。昆虫肠道微生物具有产生活性次级代谢产物的能力,是活性天然产物的重要来源。【目的】研究药用昆虫喙尾琵琶甲(Blaps rynchopetera)成虫肠道来源链霉菌(Streptomyces sp.) BPA71的次级代谢产物及其生物活性。【方法】利用正相硅胶柱色谱、葡聚糖凝胶Sephadex LH-20柱色谱等方法分离纯化该菌株的发酵粗提物,采用牛津杯法进行抗菌活性追踪,确定抗菌活性部位,通过ESI-MS、NMR等波谱数据分析对化合物结构进行鉴定,采用微量肉汤稀释法测定最低抑菌浓度(minimal inhibitory concentration, MIC),采用MTS法测定抗肿瘤活性。【结果】从Streptomyces sp. BPA71的固体发酵提取物中共分离得到4个已知化合物,通过对比核磁数据确定为糠酸甲酯(1)、吡咯甲酰胺A (2)、吡咯甲酰胺B (3)和吲哚-3-乙酸甲酯(4)。抗菌活性结果显示化合物2具有广谱抗菌活性。此外,化合物2对宫颈癌细胞HeLa、肺癌细胞A549、肝癌细胞SMMC-7721、乳腺癌细胞MDA-MB-231和结肠癌细胞SW480这5株肿瘤细胞均有明显的抑制活性。【结论】喙尾琵琶甲肠道来源Streptomyces sp. BPA71可产生丰富的生物活性物质,该研究结果为进一步挖掘喙尾琵琶甲肠道链霉菌的活性天然产物奠定了基础,同时丰富了人们对喙尾琵琶甲肠道微生物的认识。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic tissues of hybrid firs (<Emphasis Type="Italic">Abies</Emphasis> spp.) and regeneration of transgenic emblings

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l⁻¹). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l⁻¹. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.     

Relationship between DNA methylation and histone acetylation levels, cell redox and cell differentiation states in sugarbeet lines

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.A. Causevic and M.-V. Gentil contributed equally to this work.     

Activity spectra of Bacillus Thuringiensis δ-endotoxins against eight insect cell lines

Summary Eight continuous insect cell lines were tested for susceptibility to the δ-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the “lawn assay” and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.     

Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum

Summary Protoplasts were derived from haploid and diploid Nicotiana sylvestris and N. tabacum. Exposure of the protoplasts to mutagenic doses of ultraviolet (U.V.) radiation prior to two selection rounds in the presence of 4 mM (or 5 mM) and 8 mM of valine, respectively, was required to obtain cell lines with persistent valine resistance. Such lines were obtained from haploid and diploid N. sylvestris protoplasts as well as from haploid protoplasts of N. tabacum but not from (1.8 × 10⁷) diploid N. tabacum protoplasts. The ratio between number of verified valine-resistant cell lines and the initial number of U.V. exposed protoplasts enabled the estimation of the following order of mutation frequency: haploid N. sylvestris > haploid N. tabacum > diploid N. sylvestris. Plants which retained the valine resistance and transmitted it to their sexual progeny were derived from the resistant cell lines.