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.

Cytotoxicity evaluation of silica nanoparticles using fish cell lines

Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO₂ NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO₂ NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO₂ NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO₂ NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 μg/mL were needed to achieve 24 h EC₅₀ values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO₂ NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC₅₀ values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays.     

Enhanced DNA transfer into fish bone cells using polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA intoa wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.     

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.     

Transfection of cells attached to selected cell based biosensor surfaces

Mammalian cell attachment studies were conducted on a variety of common microchip surfaces for potential use in cell based biosensors. COS-7 cell attachment to Au, Pt or ITO, per unit area was greater than to SiO(2) surfaces. The number of cells that would attach was essentially maximized 3 h after cell seeding. HL-1 cells attached more readily to surfaces precoated with fibronectin, but by 3 h equivalent number of cells had attached independent of fibronectin precoating. Inclusion of serum in media during the initial period of attachment decreased the number of COS-7 cells attached to SiO(2) surfaces, but no dependence on serum was seen for ITO surfaces. The number of cells attached per unit area varied with the composition of the surface. However, no differences were observed in the percentage of cells transfected with a green fluorescent protein gene, or in the level of reporter gene expression over the population of transfected cells on ITO, SiO(2), Pt, Ag, or Au surfaces. Similar FACS analysis of transfected Hep G2 cells revealed lower levels of both transfection efficiency and levels of GFP fluorescence. Hep G2 cells plated on Ag did not remain attached for analysis, but there were no significant differences between tissue culture plastic and the other biosensor surfaces in the percentage of cells transfected. This suggests that, in general, cells will attach to the various conducting and nonconducting biosensor surfaces studied and will provide comparable data in reporter gene expression assays.     

Analysis of three marine fish cell lines by rapd assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.     

Preparation of a low molecular weight polyethylenimine for efficient cell transfection

Polyethylenimines (PEIs) of a molecular weight between 25 and about 800 kDa have successfully been used for in vitro and in vivo gene delivery approaches. Recent publications indicated that PEI molecules of lower molecular weight and a small molecular weight range are also efficient transfection reagents with a much lower cytotoxicity compared to high molecular weight PEIs. Here, we describe the application of a molecular sieve chromatography to fractionate a commercially available 25-kDa PEI. We generated three pools of PEIs with molecular weight ranges of 70-360 (I), 10-70 (II), and 0.5-10 kDa (III), respectively. We show that, in comparison with the 25-kDa PEI, pool III increased the expression of luciferase up to 100-fold and the number of transfected cells 2-3 fold. In addition, the kinetics of reporter gene expression was also much faster in pool III, compared with the 25-kDa PEI or with pools I or II. Finally, pool III showed the lowest cytotoxicity in comparison with the other PEI preparations. Thus, we provide a one-step processing of a 25-kDa PEI, resulting in a more effective and also less cytotoxic transfection reagent.     

The significance of scirrhous gastric cancer cell lines: the molecular characterization using cell lines and mouse models

Scirrhous gastric cancer (SGC) exhibits aggressiveness of the rapid infiltrating tumor cells with abundant fibroblasts. Experimental studies using SGC cell lines have obtained useful information about this cancer. Our literature search divulged a total of 18 SGC cell lines; two cell lines were established from primary SGC and the other lines were established from a metastatic lesion of SGC. Fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor-beta receptor (TβR) are linked to the rapid development of SGC. Cross-talk between the cancer cells and cancer-associated fibroblasts (CAFs) has been shown to contribute to the progression of SGC. Chemokine (C-X-C motif) receptor 1 (CXCR1) from SGC cells might be associated with the abundant CAFs in cancer microenvironments. The in vivo models established using SGC cell lines are expected to serve as a useful tool for the development of drugs such as FGFR2 inhibitors, TβR inhibitors, and CXCR1 inhibitors, which might be promising as SGC treatments. However, the number of available SGC cell lines is insufficient for the clarification of the entire biologic behavior of SGC. Since the mechanisms responsible for the characteristic aggressiveness of SGC are not fully elucidated, the establishment of new SGC cell lines could help clarify the biological behavior of SGC and contribute to its treatment.     

Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine.

Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl.3,3'-dithiobispropionimidate*2HCl (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M(w)) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the N/P ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intra-cross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.     

Induction of apoptosis by EGCG in selected tumour cell lines in vitro

One of the best recognised polyphenols of plant origin, epigallocatechin-3-gallate (EGCG) is contained mainly in green tea and in grapes. Studies performed in vivo and in vitro have demonstrated high probability of anti-neoplastic potential of the compound, due to its capacity to induce programmed cell death. The present studies were aimed at evaluation of apoptosis induction in cells of three selected tumour cell lines, subjected to action of various concentrations of EGCG. The experiment was performed on cultures of HEp-2 laryngeal carcinoma cells, LoVo colon carcinoma cells, HeLa cervical carcinoma cells and on normal myoepithelial cell line, HS. EGCG was found to induce apoptosis in cells of the examined neoplastic lines in a dose-related manner. Moreover, effect of EGCG on normal cells of HS line was found to be much less pronounced as compared to effects exerted on sensitive neoplastic cells.     

Glycine—SDS—PAGE测定小分子多肽分子量

小分子多肽分子量的测定 ,目前多采用Ｔｒｉｃｉｎｅ -ＳＤＳ -ＰＡＧＥ系统[1～ 3 ] ,但系统中的Ｔｒｉｃｉｎｅ价格昂贵 ,加之电泳时间较长 ( 3～ 4ｈ) ,为此 ,作者经过实验摸索 ,建立了Ｇｌｙｃｉｎｅ -ＳＤＳ -ＰＡＧＥ测定小分子多肽的方法。1　材料和方法表 1　制胶配方成分分离胶 (ｍｌ)浓缩胶 (ｍｌ)分离胶单体母液 1 3 3 -浓缩胶单体母液 -0 23 2ｍｏｌ/ＬＴｒｉｓ-ＨＣｌ 1 3 3 -(ｐＨ8 8)0 5ｍｏｌ/ＬＴｒｉｓ-ＨＣｌ -0 3 8(ｐＨ6 8)尿素 1 44ｇ -Ｈ2 Ｏ 0 40 910 %ＳＤＳ 40 μｌ 15 μｌ10 %ＡＰＳ 2 5 μｌ 12 μｌＴ…     

Rapid assessment of the toxicity of oil sands process-affected waters using fish cell lines

Rapid and reliable toxicity assessment of oil sands process-affected waters (OSPW) is needed to support oil sands reclamation projects. Conventional toxicity tests using whole animals are relatively slow, costly, and often subjective, while at the same time requiring the sacrifice of test organisms as is the case with lethal dosage/concentration assays. A nonlethal alternative, using fish cell lines, has been developed for its potential use in supporting oil sands reclamation planning and to help predict the viability of aquatic reclamation models such as end-pit lakes. This study employed six fish cell lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24 h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Forty-nine test water samples collected from the surface of oil sands developments in the Athabasca Oil Sands deposit, north of Fort McMurray, Alberta, Canada, were evaluated in blind. Small subsample volumes (8 ml) were mixed with 2 ml of 5× concentrated exposure media and used for direct cell exposures. All cell line responses in terms of viability as measured by Alamar blue assay, correlated well with the naphthenic acids (NA) content in the samples (R ² between 0.4519 and 0.6171; p?<?0.0001) when data comparisons were performed after the bioassays. NA or total acid-extractable organics group has been shown to be responsible for most of the acute toxicity of OSPW and our results further corroborate this. The multifish cell line bioassay provides a strong degree of reproducibility among tested cell lines and good relative sensitivity of the cell line bioassay as compared to available in vivo data that could lead to cost effective, high-throughput screening assays.     

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.     

Growth of fish cell lines in glutamine-free media

The glutamine requirement for thein vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and raibow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for thein vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement.Abbreviations BSA bovine serum albumin - CHSE-214 Chinook samon embryo cell line - dFBS dialyzed fetal bovine serum - FBS fetal bovine serum - GFSk-S1 goldfish skin cell line - GS glutamine synthetase - L-15 Leibovitz's L-15 media - L929 mouse fibroblast cell line - MEM minimum essential medium Eagle - PBS phosphate buffered saline - RTL-W1 rainbow trout liver cell line - RTSp-W1 rainbow trout spleen cell line     

Discrimination between selected lines of pigs using AFLP markers

Amplified fragment length polymorphic (AFLP) markers were used to discriminate between lines of pigs, divergently selected over seven generations for components of efficient lean growth rate. A total of 270 animals with 30 animals per line were genotyped for 239 polymorphic AFLP markers. Canonical variate analysis identified linear combinations of the AFLP marker scores that grouped animals by selection line with no overlap between selection lines. Cluster analysis of AFLP marker scores identified 10 groups of animals with 226 of the 270 animals clustered into nine groups, each consisting of animals from only one selection line. AFLP marker genotyping, using the EcoRI and TaqI restriction enzymes, provided an effective means of discriminating between animals of different selection lines that have arisen from one base population.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Comparison of two birnavirus-rhabdovirus coinfections in fish cell lines

Aquabirnaviruses, such as the infectious pancreatic necrosis virus (IPNV), Novirhabdoviruses, such as the infectious hematopoiteic necrosis virus (IHNV) and the viral hemorrhagic septicemia virus (VHSV), cause considerable losses to the salmonid industry worldwide. Coinfections of 2 viruses have been described, but the interactions between rhabdoviruses and birnaviruses have not been examined closely. Using virus titration, flow cytometry and RT-PCR assays, we compared the effect of IPNV on the replication of IHNV and VHSV in tissue culture cells. RT-PCR assays indicated that simultaneous infection of IPNV with VHSV does not affect the replication of the rhabdovirus either in the first or successive passages; the infective titers were similar in single and double infections. In contrast, coinfection of IPNV with IHNV induced a fall in infectivity, with reduced expression of IHNV viral antigens in BF-2 cells from Lepomis macrochirus and a loss of 4.5 log10 units of the infective titer after 3 successive passages. It was possible to stimulate BF-2 cells to produce significant interferon-like activity against IHNV but not against VHSV.