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.     

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.     

Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer

For the first time gene cloning systems have been developed for Amycolatopsis japonicum. Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A. japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer. The direct transformation procedure was modified to introduce DNA. The most important parameter for an efficient DNA uptake was the age of the culture. Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies. Further, conditions for transformation of A. japonicum protoplasts were established. The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar. The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation. The plasmid was genetically stable, and could easily be reisolated from A. japonicum. We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible. The plasmid pSET152 integrated in the A. japonicum chromosome. A titre of 2.4 x 10(-4) exconjugants per recipient was obtained.     

Chiral multinuclear macrocyclic polyamine complexes: synthesis, characterization and their interaction with plasmid DNA

A series of multinuclear macrocyclic polyamine metal (Zn(2+), Cu(2+), Co(2+)) complexes containing chiral dipeptide linkage were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and plasmid DNA (pUC19) was studied, and the results revealed that these complexes could act as powerful catalysts for the cleavage of plasmid DNA under physiological conditions.     

PEG-assisted DNA solubilization in organic solvents for preparing cytosol specifically degradable PEG/DNA nanogels

DNA was dissolved in selected organic solvents in the presence of poly(ethylene glycol) (PEG). Nanoscale PEG/DNA complex (approximately 100 nm) was produced in dimethylsulfoxide (DMSO) phase. Using a thiol-functionalized six-arm branched PEG for DNA solubilization, the PEG/DNA nanocomplex was cross-linked through the formation of disulfide linkages between the thiol groups, resulting in the production of stable PEG/DNA nanogels in aqueous solution. DNA release from the nanogels could be modulated by changing the concentration of an external reducing agent. The released plasmid DNA from the nanogels maintained intact structural integrity and exhibited appreciable gene transfection efficiency. The PEG/DNA nanogels could be potentially applied for gene therapy including DNA vaccination.     

PAMAM-PEG-PAMAM: novel triblock copolymer as a biocompatible and efficient gene delivery carrier

A novel triblock copolymer, PAMAM-block-PEG-block-PAMAM was synthesized and applied as a gene carrier. PAMAM dendrimer is proven to be an efficient gene carrier itself, but it is associated with certain problems such as low water solubility and considerable cytotoxicity. Therefore, we introduced PEG to engineer a nontoxic and highly transfection efficient polymeric gene carrier because PEG is known to convey water-solubility and biocompatibility to the conjugated copolymer. This copolymer could achieve self-assembly with plasmid DNA, forming compact nanosized particles with a narrow size distribution. Fulfilling our expectations, the copolymer was found to form highly water-soluble polyplexes with plasmid DNA, showed little cytotoxicity despite its poor degradability, and finally achieved high transfection efficiency comparable to PEI in 293 cells. Consequently, these data show that an approach involving the introduction of PEG to create a tree-like cationic copolymer possesses a great potential for use in gene delivery systems.     

Transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA

Abstract A method for efficient polyethylene glycol (PEG)-mediated transformation of Bacillus amyloliquefaciens protoplasts with plasmid DNA is described. The best conditions found for protoplast regeneration included using 0.45 M sucrose both during the cultivation of the cells and (as an osmotic stabilizer) during their treatment with lysozyme, whereas 0.25 M sodium-succinate was added to the regeneration plates. Under these conditions about 5–10% of input cells regenerated. The highest transformation frequency with plasmid DNA was obtained with a PEG 6000 concentration of 22.5% (w/v). Transforming B. amyloliquefaciens strains with the plasmid pUB110 isolated from B. amyloliquefaciens resulted in 2–4 · 10⁵ transformants/μg DNA, 100–1 000-times as high as with DNA from Bacillus subtilis , suggesting a restriction barrier between the two species. Transformation of B. amyloliquefaciens with plasmids pC194 or pE194 cop -6 gave poor yields and no restriction barrier could be demonstrated for these plasmids. However, by curing pC194 from one of the transformants, a mutant strain compatible to both the plasmids could be isolated, yielding 2–3·10⁴ transformants/μg DNA. Both laboratory and industrial B. amyloliquefaciens strains could be transformed with the procedure.     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.     

Factors affecting PEG-mediated stable transformation of maize protoplasts

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 11 ratio to 65% at a 1001 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.Abbreviations PEG polyethylene glycol - BMS Black Mexican Sweetcorn - MS salts of Murashige and Skoog (1962) culture medium - MaCa 0.2M mannitol, 80mM calcium chloride - MaMg 0.4M mannitol, 15mM magnesium chloride, 0.1% MES, pH 5.6 - CM conditioned medium - SE standard error of the mean     

PEG介导的外源基因对绿豆叶肉原生质体的转化和瞬间表达研究(简报)

作者曾利用Krens等(1982)的PEG法将NPT-Ⅱ丛因直接导入绿豆原生质体并获得稳定表达,但该法的转化率极低。为了建立绿豆原生质体有效的转化系统,我们将PEG法在大豆原生质体转化的基础上进一步改进,利用GUS基因为报告基因,对三个不同品种的绿豆叶肉原生质体进行直接转化。并对影响PEG转化的有关因素进行了研究。     

PEG-mediated plastid transformation: a new system for transient gene expression assays in chloroplasts

Summary Evidence is presented for the introduction of functional copies of the GUS-reporter gene with plastid regulatory signals into chloroplasts after treatment of Nicotiana plumbaginifolia leaf protoplasts with PEG. GUS-activity is found in cells derived from protoplasts treated with PEG in the presence of plasmids harbouring the GUS-gene under the control of plastid promoter and terminator signals (plastid-specific reporter gene constructions). The activity is maintained after chloroplast isolation and incubation with the protease thermolysin under conditions sufficient to completely remove the much higher transient nuclear/cytoplasmic expression of a GUS-gene carrying the CaMV 35S-promoter. Likewise, GUS-activity derived from a plasmid coding for the nuclear/cytoplasmic expression of the reporter gene with a plastid transit presequence is also maintained after these procedures. These results indicate that PEG-treatment is a suitable protocol by which to introduce DNA into chloroplasts for the study of transient gene expression.     

Synthesis of a novel linear polymer of a macrocyclic polyamine copper (II) complex and its interaction with plasmid DNA

The novel linear polymer of a macrocyclic polyamine copper (II) complex, which has many cyclen groups linked by epichlorohydrin, has been synthesized as a DNA cleavage agent. The structure of the polymer 3 was identified by 1HNMR and IR and its molecular weight was measured by GPC. The result of agarose gel electrophoresis assay showed that Cu-(II) complex 4 could act as a powerful catalyst for the cleavage of plasmid DNA under physiological conditions.     

Ternary Interpolyelectrolyte Complexes DNA–Polycation–Polyanion: A New Approach to Optimization of Mixtures for Transfection of Eukaryotic Cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M30–40 kDa as polycation and polyacrylic acid (PA) with M20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA–PEG incorporation into a ternary complex (by itself or as a 1 : 1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA–PEG (as a 1 : 9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a -galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1 : 1 mixture of PA and PA–PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Glycine-dependent cryotransformation of Bacillus anthracis by DNA of various plasmids

The possibility of the 1,8-60,0 Md plasmids DNA transfer into the cells of different Bacillus anthracis strains has been established. A number of parameters of the procedure has been optimized. The including of PEG6000 into the cryomixture in the final concentration 10% has been demonstrated to be vital for increase in transformants yields. Under the described conditions the maximal efficiency of transformation (1,2.10(3)) has been registered for the DNA of the plasmid pUB110. The efficiency of the process decreased concomittant with the size of the transformed plasmids representing 2-4.10(-1) for the plasmid perlicon pXO2 (60 Md). The plasmids obtained by Bacillus anthracis cells retained the functional and structural stability.