A simple method for decreasing the toxicity of polyethylene glycol in mammalian cell hybridization.

The yield of hybrid colonies after fusion of mammalian cells with polyethylene glycol (PEG) is increased if the cells are fused in Ca2+-free medium, and kept in Ca2+-free medium for at least 15 min after fusion. The protective effect of Ca2+-free medium is much more obvious when Baker PEG is used than when fusion is carried out with Koch-Light PEG. The increased yield of hybrid colonies is shown to be due to a reduced toxicity rather than to an increased efficiency of cell fusion. These improvements have been found to apply to a variety of cell lines, and also when cell fusion is carried out in suspension. This technique should be particularly useful in studies on mammalian cell hybridization using cell lines that are particularly sensitive to the toxic effect of PEG.     

Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400-6000 was found to occur in the concentration range of 50-55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.     

A new dimension in suspension fusion techniques with polyethylene glycol.

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as "pancake fusion," is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG-treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all-or-none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.     

Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents

Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG₁₂-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate.     

Introduction of Foreign Gene into Small Cell Groups Digested Partial-Enzymically in Rice and Regeneration of Transgenic Plants

Suspension cultures of small cell groups (SCG; ca. 50–100 cells per group) were established from calli of Japonica rice Fang 7 and Hl24. The SCG were partially digested and transformed by plasmid pBll21 harboring the NPT-II (neomycin phosphotransferase) and GUS (betaglucuronidase) genes. Plasmid DNA was introduced into cells' by PEG, electroporation and PEG plus electroporation. NTP-II and GUS activity assay showed that the report genes were expressed in transformed cells. Transgenic plants were regeneiated possessing GUS activity due to the integration of intact foreign DNA into their genome as evidanced by hybridization. The results prove that the partially digested SCG is a potential, feasible system as receptor for gene transfer, especially for plants which are difficult for protoplast culture and plant regeneration from protoplasts.     

难免流产蜕膜组织遗传印记基因PEG10的表达

采用半定量逆转录聚合酶反应(RT-PCR)、原位杂交、免疫印记(Western blot)及免疫组织化学技术检测了36例难免流产患者蜕膜组织PEG10 (Paternally expressed gene 10) mRNA及蛋白的表达与分布, 并以36例同期正常早孕妇女为对照, 研究遗传印记基因在难免流产蜕膜组织中的表达, 探讨其在自然流产中的作用。RT-PCR结果显示, PEG10在两组蜕膜组织中均有表达, 正常妊娠组平均表达水平为0.5994±0.049, 难免流产组为0.1783±0.037, 两组比较具有显著性差异(P<0.05)。原位杂交、免疫组化及Western blot分析也显示PEG10的表达规律与RT-PCR结果相吻合。研究结果表明, 遗传印记基因PEG10维持一定水平的表达对早期胚胎发育和正常妊娠的维持有重要意义, 而其表达下调可能是导致难免流产的原因之一。     

Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA

We developed a facile method for plasmid DNA (pDNA) extraction from crude Escherichia coli lysate using magnetic hydroxyapatite nanoparticles (MHapNPs) in the presence of polyethylene glycol (PEG)/NaCl. DNA condensation induced by PEG/NaCl is a prerequisite for achieving pronounced DNA recovery. The quality and quantity of MHapNP-purified pDNA under optimal binding buffer conditions (0.5 volume of 20% PEG 8000/2M NaCl) were comparable to those obtained using organic solvents or commercial kits. This MHapNP technique is rapid, simple, cost-effective, and environmentally friendly and has the potential to extract DNA from other cell lysates.     

Yeast cell debris and protein partitioning in the poly(ethylene glycol)-poly(vinyl alcohol) biphasic system

Yeast cells, cell debris and protein partitioning have been investigated in the poly(ethylene glycol) (PEG) 8000/poly(vinyl alcohol) (PVA) 10,000 system. Cells and cell debris partition into the lower (PVA) phase over the pH range 4.8-7.5, and with up to 0.37 M KCl at pH 5.9. Protein partitioning is more pH-dependent in the PEG/PVA system than in the PEG/dextran system, and a significant fraction of the total protein is found at the interface at lower pH values. Significant, rapid purification of overproduced pyruvate kinase in a PEG/PVA system containing Blue Sepharose CL-6B particles is demonstrated.     

Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization

A rapid method for localizing large numbers of complete cosmids by nonradioactive in situ hybridization is described. The cosmids are nick translated in the presence of biotin-16-dUTP, incubated with an excess of sonicated human DNA, and used as a probe for in situ hybridization. Sites of hybridization are detected by successive treatments with FITC-labeled avidin and biotinylated anti-avidin antibody. Fifty-two cosmids were localized on chromosome 16 in 5 d relative to translocation breakpoints contained in two cell lines. Rapid identification of chromosome 16 was achieved by cohybridization with a chromosome 16-specific centromeric repeat probe.     

Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides.

Polyethylene glycol (PEG)-coupled oligonucleotides are partitioned in an aqueous two-phase system PEG/dextran. The affinity of the oligonucleotide for the PEG-rich phase increases proportionally to the length of the coupled PEG polymer. After hybridization, the PEG-coupled oligonucleotide is able to force a complementary nucleic acid strand into the PEG-rich phase. This property can be used for the sequence-specific isolation of nucleic acids through hybridization-based affinity partitioning. The dependence of the partition coefficient in this system on various parameters is described. The application of this principle to multistage chromatographic separations is demonstrated.     

白花柽柳质膜水孔蛋白基因克隆及序列分析

植物水孔蛋白在植物体内形成水选择性运输通道,在植物种子萌发、细胞伸长、气孔运动、受精等过程中调节水分的快速跨膜运输。有的水孔蛋白还在干旱胁迫应答中起重要作用。本文根据白花柽柳的PEG6000胁迫处理构建的SSH消减文库的水孔蛋白基因表达序列标签(EST),设计基因特异性引物进行5′RACE,克隆出一个1 043 bp的核苷酸序列。应用生物信息学软件进行分析,预测该序列编码287个氨基酸,具有6个跨膜区,有MIP家族信号序列SGXHXNPAVT,高等植物PIP高度保守序列GGGANXXXXGY和TGI/TNPARSL/FGAAI/VI/VF/YN,这是质膜水孔蛋白基因的典型的结构特征。经NCBI比对,与Arabidopsis thaliana (MIP-C),同源性达到95%,预测该蛋白的相对分子量是30.9KD,理论等电点是8.84。     

Fibroblast aggregation by suspension with conjugates of poly(ethylene glycol) and RGD

Cell aggregates may be useful components of artificial organs and mammalian cell bioreactors, but many cells do not naturally aggregate. In a previous report,(4) we described a method for promoting neural cell aggregation by addition of water-soluble conjugates of cell adhesion peptides, containing the three amino acid sequence Arg-Gly-Asp (RGD), and poly(ethylene glycol) (PEG). Here, we examined the mechanism of conjugate-induced aggregation using fibroblasts and a variety PEG-peptide conjugates. Aggregation was monitored during rotation culture of fibroblasts in the presence of unconjugated GRGDY and PEG; monofunctional (PEG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctional conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY). GRGDY-PEG-GRGDY conjugates induced rapid and pronounced fibroblast aggregation that was dose-dependent; at the highest concentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were produced more quickly ( approximately 1 h) and were significantly larger at 24 h (mean radius approximately 66 mum) than at slightly lower concentrations (1.7 and 3.3 mg/mL). Aggregation with GRGDY-PEG-GRGDY was completely inhibited by dissolved GRGDY (1.7 mg/mL). Neither unmodified GRGDY, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to significant aggregation. The extent of aggregation depended on PEG molecular weight: conjugates with 3400 M(w) PEG produced aggregates with significantly larger mean radius than conjugates with 20,000 M(w) PEG. When 1N-8A fibroblasts, genetically engineered to produce recombinant nerve growth factor (NGF), were aggregated with GRGDY-PEG-GRGDY, aggregated cells produced more NGF per cell than nonaggregated cells. Aggregation of cells may lead to improved cell function, such as the increase in NGF production observed here, which could be useful in large-scale cell culture and construction of artificial organs or tissue transplants for tissue engineering. (c) 1996 John Wiley & Sons, Inc.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

Optimized immobilization of activated sludge in poly(ethylene glycol) gels by UV technology

Conventional methods for immobilization of microorganisms in hydrogel are not very efficient and cost-effective. Ultraviolet (UV) technology, a rapid method for hydrogel preparation in ambient environment, was applied to immobilize activated sludge in poly(ethylene glycol) (PEG) gel carriers synthesized from PEG pre-polymer. Conditions for cell immobilization were optimized by selecting suitable UV photoinitiators, crosslinker concentrations, and protection gas. The optimum conditions are the following: 12% PEG pre-polymer with 1% MBA and 0.1% DEAP. More than 90% of ammonia was reduced, and 75% of the total organic compound was removed after treating 40 and 10 mg/L synthetic ammonia wastewater with different C/N ratios in 5 h under aeration conditions. Ammonia degradation increased with increasing C/N ratios, and the scanning electron microscopy images revealed that the immobilized beads had porous structures dominantly filled with nitrifiers. The bioactivity of the immobilized activated sludge was maintained during immobilization by UV technology.     

Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes

The mechanism by which polyethylene glycol (PEG) mediates cell fusion has been studied by examining the movements of membrane lipids and proteins, as well as cytoplasmic markers, from erythrocytes to monolayers of cultured cells to which they have been fused. Fluorescence and freeze-fracture electron microscopy and fluorescence recovery after photobleaching have yielded the following results: (a) In the presence of both fusogenic and nonfusogenic PEG membranes are brought together at closely apposed contact regions. (b) Fluorescent lipid probes quickly spread from the membranes of erythrocytes to cultured cells in the presence of both fusogenic and nonfusogenic PEG. (c) Proteins of the erythrocyte membranes were never observed to diffuse into the cultured cell membrane. (d) Water-soluble proteins did not diffuse from the erythrocyte interior into the target cell cytoplasm until the PEG was removed. These data suggest that the coordinate action of two distinct components is necessary for fusion as mediated by PEG. Presumably, the polymer itself promotes close apposition of the adjacent cell membranes but the fusion stimulus is provided by the additives contained in commercial PEG.     

Time and cell systems as variables in fusion experiments with polyethylene glycol

Summary Cell hybridization was done between a monolayer of B14-150 Chinese hamster cells and a suspension of either mouse leukemia cells or normal human lymphocytes. Cell contact was obtained by centrifugation of the suspension cells onto the monolayer cells in a culture plate. Cell fusion was done by means of polyethylene glycol (PEG). The optimum time for PEG exposure as well as the yield of hybrid cells differed markedly with the different combinations.     

A reproducible microanalytical method for the detection of specific RNA sequences by dot-blot hybridization

A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.