Plasmid DNA extraction by double magnetic beads method and its application / 生物工程学报

Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.

Research progress on stable gene transfection methods of Plasmodium falciparum / 中国热带医学

@#Abstract: Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance. However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure (A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection (LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.

Development and applications of quality control materials for detection of CYP3A5 genotypes / 临床检验杂志

Objective@#To evaluate the external quality assessment (EQA) program for genotyping results of tacrolimus metabolism-related cytochrome P450 family 3 subfamily A member 5 ( CYP3A5 )using plasmid DNA constructed in vitro as quality control samples, discuss the problems in clinical laboratories enrolled in the program and improve the detection quality of CYP3A5 gene. @*Methods@#Recombinant plasmid carrying CYP3A5 *3 (rs776746) AA locus sequence was constructed as wild type sample and plasmid with CYP3A5 *3 GG mutation as mutant type sample. Heterozygous mutant samples were obtained by mixing the two plasmids with equal proportion. Recombinant plasmids DNA were used as the sample panel for EQA scheme. Participating laboratories were asked to test the samples using their routine methods and report the results before deadlines. The scores of each laboratory were calculated based on their results and the overall coincidence of different samples as well as the sensitivity and specificity of different methods. @*Results@#CYP3A5 *3 locus genotypes of the constructed plasmid were verified by Sanger sequencing. The results of 15 and 17 valid laboratories were received respectively in the two EQA programs. The total percentage of 93.33% (14/15) and 100% (17/17) of the laboratories submitted correct results for all the samples. The overall coincidence rates were 96% (72/75) and 100% (85/85) respectively. All the laboratories using digital FISH got full marks in two EQA schemes, while the coincidence rates were 90% (27/30) and 100% (40/40) for Sanger sequencing. @*Conclusion@#The recombinant plasmid DNA constructed in this study could effectively detect the performance of reagents with good clinical applicability. The results of EQA programs suggested that the overall accuracy rate of enrolled laboratories was high enough, while the performances in some laboratories still need to be improved. Quality controls in clinical laboratories were essential to assure the accuracy of results.

Preparation and in vitro evaluation of biodegradable polypeptide gene vector / 第二军医大学学报

Objective To prepare a lipoic acid modified polyarginine polypeptide nanocomplex for gene delivery system, and to observe its transfection efficiency and cytotoxicity on HEK293 cells. Methods We synthesized four disulfide cross-linked lipoic acid modified polyarginine peptide and histidine (LHRss) at different cross-linked degrees using different mol fraction of L-cysteine as cross-linking agent. The construction of LHRss was characterized by1H nuclear magnetic resonance (1H NMR) and gel permeation chromatography. The LHRss/plasmid DNA (pDNA) nanocomplexes were self-assembled with LHRss and pDNA at different nitrogen/phosphorus (N/P) ratios. The size and zeta potential of LHRss/pDNA nanocomplexes were characterized by particle size analyzer, and the pDNA enrichment capability was determined by electrophoretic mobility shift assay (EMAS). Then, the intracellular uptake and gene transfection efficiency of LHRss/pDNA nanocomplexes in HEK293 cells were investigated. CCK-8 method was used to determine the cytotoxicity of LHRss/pDNA nanocomplexes on HEK 293 cells. Results1H NMR results showed that LHRss was successfully synthesized. The nanocomplexes had a uniform distribution of particle size, and the zeta potential of LHRss3/pDNA and LHRss4/pDNA nanocomplexes were more than 30 mV when N/P≥40. EMAS results showed that pDNA could be completely wrapped by LHRss3 when N/P=5. When N/P=40, the intracellular uptake and transfection efficiency of LHRss3/pDNA nanocomplex by HEK293 cells was significantly higher than that of other three nanocomplexes and lipoic acid modified polyarginine peptide and histidine (LHR)/pDNA; the mean fluorescence intensity of LHRss3/pGL3 nanocomplexes was approximately 3. 98 times that of the LHR/pGL3 nanocomplex (P<0. 05). Cytotoxicity results showed that the cell survival rates were more than 80% at 24 h after transfected with LHR/pGL3 and LHRss/pGL3, and its toxicity was significantly lower than that of bPEI-25K, with the cell survival rates being about 25% at 24 h after transfected with 20 μg/mL bPEI-25K (P<0. 05). Conclusion The prepared lipoic acid modified polyarginine polypeptide nanocomplex is expected to become an efficient gene vector.

Fluorescence analysis of interaction between 5 alkaloids from Rhizoma Coptidis with protein and DNA / 第二军医大学学报

Objective: To study the interaction between biomacromolecules (protein and DNA) and different Rhizoma Coptidis alkaloids (palmatine, coptisine, epiberberine, jatrorrhizine and berberine) and the difference among 5 Rhizoma Coptidis alkaloids. Methods: Bovine serum albumin (0.5 mL of 1.10 × 10-5 mol/L) was added to 1.5mL of each alkaloid of different concentrations (concentrations of palmatine, coptisine, epiberberine, jatrorrhizine and berberine being 0, 1.95×10-6, 3.9×10-6, 7.8×10-6, 1.56×10-5 and 3.12×10-5 mol/L, respectively). The reaction system was shaken and incubated at 37°C for 1h, and then the emission spectra of mixed solution were recorded within 300 to 500nm at 280nm excitation. Plasmid DNA (0.5mL of 0.1%) was added to 1.5mL of each alkaloid with different concentrations (concentrations of palmatine, coptisine, epiberberine, jatrorrhizine and berberine being 0, 1.95×10-6, 3.9×10-6, 7.8×10-6 and 1.56×10-5 mol/L, respectively). The reaction mixture was well shaken, and then the fluorescent emission spectrum was recorded from 480 to 650 nm at 368 nm excitation by an F-4500 fluorescence spectrophotometer. Results: All the Rhizoma Coptidis alkaloids in test exhibited similar quenching effect on fluorescence of bovine serum albumin, and they all showed fluorescence enhancing effect on plasmid DNA, and the enhancing effects varied greatly among the five alkaloids, with epiberberine showing the strongest activity, followed by coptisine, palmatine, berberine, and jatrorrhizine in order. The strongest interaction between DNA and five alkaloids were found at the concentration of 7.8×10-6mol/L. Conclusion: Strong interaction can be found between Rhizoma Coptidis alkaloids and biomacromolecules including protein and DNA. The interaction of Rhizoma Coptidis alkaloidswith plasmid DNA varies greatly among different alkaloids and is similar with bovine serum albumin. 9, 10-methylene-dioxy groups and 2, 3-dimethoxy groups might have promoted the interaction between alkaloids and DNA.

Optimized preparation of DNA-chitosan nanoparticles with high transfection effi-cency through a central composition design / 药学实践杂志

Objective This study aimed to optimize the preparation condition of DNA-chitosan nanoparticles with high transfec-tion efficency through a central composition design .Methods The DNA-chitosan nanoparticles were prepared by complex coacervation between pEGFP and chitosan .We selected the concentrations of chitosan and plasmid as two experimental factors , and a central compos-ite design with two factors and five levels was used to optimize the preparation condition of DNA-chitosan nanoparticles for high transfec-tion efficency .The concentrations of chitosan and plasmid were selected as the independent variables , respectively .The dependent varia-bles included average particle size and transfection efficiency .The morphology of DNA-chitosan nanoparticles was observed using a trans-mission electron microscope .The size and zeta potential of nanoparticles were measured by dynamic light scattering ( DLS) and electro-phoretic light scattering ( ELS ) , respectively .The stability of plasmids in the process of nanoparticles preparation was investigated through the agrose gel electrophoresis .The expression of plasmids delivered by nanoparticles was observed under an inverted fluorescence microscope .The transfection efficiency of DNA-chitosan nanoparticles was assayed by flow cytometry .Results The preparation condition of DNA-chitosan nanoparticles with high transfection efficency was optimized successfully .Under the optimum preparative conditions , the DNA-chitosan nanoparticles were almost spherical .The average size of nanoparticles was 217.6nm, and distributed in a narrow range with a polydispersity index of 0.241.The zeta potential was +22.4 mV, which suggested that a den-sity of positive charge exist onto the surface of nanoparticles and consequently enhanced the stability of nanoparticles suspension .The results of gel electrophoresis showed that plasmids were not destroyed in the process of nanoparticles preparation .The cell transfection of nanoparticles was very highly efficient .The nanoparticles could effectively deliver the pEGFP plasmids into cells to express the green fluorescent protein at a high level.Conclusion The established mathematic models have the good predictive function .Under the optimum preparative condi-tions, the DNA-chitosan nanoparticles have the high potential of cell transfection .

Study on gene delivery system based on bisphosphonate-mediated gene-eluting stent / 国际生物医学工程杂志

ObjectiveThe aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immuno-conjugation for achieving site-specific gene delivery.MethodsA gene eluting stent was fabricated by reacting with polyallylamine bisphosphonate (PAA-BP) to introduce amine reactive groups on the surface.Then an anti-DNA antibody was chemically coupled and pDNA was immunologically tethered on the stent surface.Radioactive-labeled antibody was used to evaluate binding capacity and stability.ResultsThe presence of amine groups on the modified stent surface was confirmed by XPS and AFM analysis.The isotope label assay indicated that the amount of antibody chemically linked on the stents was 15-fold higher than that of the control stent and its retention time was also significantly longer.ConclusionThe results suggested that a large amount of reactive amine groups were introduced on the PAA-BP modified 316L coronary stent surface.This study provide a potential metal surface modification method that could facilitate coupling and tethering of biological molecules such as anti-DNA antibody and plasmid DNA (pDNA) to achieve sustained and highly localized gene delivery for substrate-mediated gene transfection.

Polymeric Gene Delivery for Diabetic Treatment

Several polymers were used to delivery genes to diabetic animals. Polyaminobutyl glycolic acid was utilized to deliver IL-10 plasmid DNA to prevent autoimmune insulitis of non-obese diabetic (NOD) mouse. Polyethylene glycol grafted polylysine was combined with antisense glutamic acid decarboxylase (GAD) MRNA to represent GAD autoantigene expression. GLP1 and TSTA (SP-EX4) were delivered by bioreducible polymer to stop diabetic progression. Fas siRNA delivery was carried out to treat diabetic NOD mice animal.

Development of potency assays for a plasmid containing vascular endothelial growth factor 2 / Desarrollo de la potencia de los ensayos para un plásmido que contiene factor 2 de crecimiento endotelial vascular

We have developed analytical methods to measure the biological functions of pVGI.1(VEGF2), a naked plasmid DNA product containing vascular endothelial growth factor 2 used in clinical trials for coronary artery diseases (CAD) and peripheral artery diseases (PAD). After being injected into muscles, vascular endothelial growth factor 2 (VEGF-2), presumably expressed in muscle tissues, binds to the endothelial cell receptors VEGFR2 or VEGFR3, triggering the downstream responses including cell proliferation and vascularization. As it is important to make sure clinical material is biological active, we developed a quantitative assay first to measure the receptor binding activity of the pVGI.1(VEGF2) gene product expressed by the transfected host cells, and then a qualitative assay to confirm the cell proliferation promoting activity of the expressed protein. In both assays the signals were plotted directly against input DNA concentrations used to transfect the host cells. We confirmed specificity for both assays. In addition, we demonstrated acceptable levels of spike recovery (86.7-116 percent), precision (largest relative standard deviation (RSD)=19.3 percent), linearity and range (60-140 percent relative potency, 15 - 35 ug/mL) for the quantitative assay. We intend to use the potency assays for routine lot release and stability studies.

Inhibitory effects of interleukin-10 plasmid DNA on the development of atopic dermatitis-like skin lesions in NC/Nga mice

Interleukin (IL)-10 exerts potent anti-inflammatory effects by suppression of both T-help (Th) 1 and Th2 cells. Previous studies have reported that IL-10 can ameliorate various inflammatory disorders. The present study was performed to examine whether IL-10 plasmid DNA could suppress development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice, as an initial step towards the development of an appliance for use in dogs with AD. Intradermal injection of IL-10 plasmid DNA markedly inhibited the development of AD-like skin lesions, as evidenced by a marked decrease in skin symptoms and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Moreover, relative mRNA expression levels of IL-4 and interferon-gamma in the skin lesions of mice injected with IL-10 plasmid DNA were also decreased compared with those of control mice. Of note, higher serum IL-10 levels in mice injected with IL-10 plasmid DNA were maintained compared with those in control mice. Taken together, the results indicate that IL-10 plasmid DNA can suppress the development of AD-like skin lesions by suppressing both Th1 and Th2 cell responses. Beneficial effects of IL-10 plasmid DNA may be expected in dogs with AD.

Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

The efficiencies of different transformation methods of E. coli DH5α strain, induced by several cations like Mg2+, Mn2+, Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

Inducible IL-12 gene expression mediated by naked DNA containing an RU486 regulatory system / 中华微生物学和免疫学杂志

Objective To evaluate the inducibility of target gene expression induced by plasmid DNA carrying a RU486 regulatory system. Methods Plasmid pRS-LacZ encoding LacZ gene and plasmid pRS22 encoding murine interleukin-12(IL-12)gene were used to bansfect cells in vitro or be rapidly injected into the vein of mice. Then LacZ expression was assayed in cultured cells and tissues by X-gal staining.The IL-12 level was tested in the supernatants of cultured cells and in the serum with ELISA. ResultsIn the presence of RU486.30％ of the liver derived hepatic HepG2 cells were dyed blue while less than 1％of the HepG2 cells were blue colored in the absence of RU486. Less than 0.5％ of other non-hepatic cells were in blue with or without RU486. After transfection, the IL-12 level in the supernatants of HepG2 cells increased with increasing concentrations of RU486 treatment. After intravenous injection of plasmid pRS-LacZ,blue colored cells were observed only in liver tissue while induced by RU486. After intravenous injection of plasmid pRS22,the IL-12 expression level in serum was related with the dosage of RU486 or/and plasmid DNA. Conclusion Plasmid DNA containing RU486 regulatory system could open or close IL-12 gene expression by the addition or deletion of RU486. And the IL-12 expression level in serum could be regulated by altering the dose of plasmid DNA or/and RU486.

A Chrysobalanus icaco extract alters the plasmid topology and the effects of stannous chloride on the DNA of plasmids

Chrysobalanus icaco (C. icaco) leaves are used in folk medicine (known as Abajeru in Brazil) to control the glycaemia in diabetic patients. Stannous chloride (SnCl2) is a powerful reducing agent used for different purposes and presents cytotoxic and genotoxic effects. The aim of this work was to investigate the effect of an aqueous C. icaco extract on the plasmid DNA topology and on the effects of the stannous chloride on DNA plasmid. Plasmid pBSK was incubated with a C. icaco extract in the presence or absence of SnCl2 (200 mg/mL), after that, the agarose gel electrophoresis procedure was carried out. Plasmid incubated only SnCl2 was used as positive control and, as negative control, plasmid incubated with Tris buffer. The gels were stained with ethidium bromide, DNA bands were semiquantified by densitometry. The data showed that C. icaco extract alters the electrophoretic profile and decreases significantly (p < 0.05) the effect of SnCl2 on plasmid DNA. The results obtained in this work could indicate a dose-dependent protective action and a genotoxic effect of C. icaco extract on plasmid DNA.

Folhas de Chrysobalanus icaco (C. icaco) são usadas na medicina popular (conhecido como Abajeru no Brasil) para controlar a glicemia em pacientes diabéticos. Cloreto estanoso (SnCl2) é um agente redutor potente usado para diferentes propostas e apresenta efeitos citotóxico e genotóxico. O objetivo deste trabalho foi investigar os efeitos de um extrato aquoso de C. icaco na topologia de DNA plasmidial e nos efeitos do cloreto estanoso sobre o DNA plasmidial. Plasmídios pBSK foram incubados com um extrato de C. icaco na presença ou ausência do SnCl2 (200 mg/mL), em seguida, o procedimento de eletroforese em gel de agarose foi realizado. Plasmídios incubados somente com SnCl2 foram usados como controle positivo e, como controle negativo, plasmídios incubados com tampão Tris. Os géis foram corados com brometo de etídio e as bandas de DNA foram semiquantificadas por densitometria. Os dados mostraram que o extrato de C. icaco altera o perfil eletroforético e diminui significativamente (p < 0,05) os efeitos do SnCl2 sobre DNA plasmidial. Os resultados obtidos neste trabalho indicam uma ação protetora dependente da dose e um efeito genotóxico de extrato de C. icaco sobre o DNA plasmidial.

Effect of Foreign Plasmid DNA on Material and Energy Metabolism of Spleen in Mice / 中国生物工程杂志

To study the influence of foreign plasmid DNA on spleen metabolism in immune-stimulated mice via the gastrointestinal tract. Mice were oral administered by pipette with 200?g of plasmid pcDNA3 and spleen were isolated at 4h and 18h after oral administration. Total RNA were extracted from spleen and spleen gene expression profile of Balb/c mice was analyzed by using Affymetrix oligonucleotide genechip after oral plasmid pcDNA3 administration. Functional cluster analysis was conducted by Genmapp and MAPPFinder software. By functional cluster analyzing the genes which were up-regulated more than twofolds, Genmapp results showed that plenty of metabolic pathway were induced in spleen after oral administration of plasmid pcDNA3. These metabolic process included purine metabolism, pyrimidine metabolism, protein synthesis, cholesterol synthesis, fatty acid synthesis, Glycolysis, TCA cycle and mitochondria oxidative phosphorylation pathway. The similar results also took place at 18h after oral administration. The result indicated that foreign plasmid DNA can modulate metabolism process in spleen of mice via the gastrointestinal tract, and may help understand the mechanism of action of foreign plasmid DNA uptaked via the gastrointestinal tract.

Trojan Peptides Based Nucleic Acids Delivery / 医学研究杂志

Trojan peptides,also named cell-penetrating peptides or protein transduction domains,are a class of small cationic peptides that often contains less than 30 amino acids.They can deliver a wide range of "cargos" such as peptides,proteins and nucleic acids efficiently through the cellular membrane.This review mainly focuses on the recent progress on utilizing Trojan peptides to deliver plasmid DNA and siRNA into cells in vitro and in vivo,and also highlights the implications of this technology in both gene function study and therapeutic potential.

A Comparative Study of Intra-arterial and Intramuscular Administration of phVEGF165 in a Chronic Ischemic Model of Rabbit Hindlimb

PURPOSE: To obtain phVEGF165 for angiogenesis and to compare the effects of its intra-arterial and intramuscular administration in a chronic ischemic rabbit hindimb model. MATERIALS AND METHODS: Chronic ischemic models were constructed in the left hindlimb of rabbits and divided into control (n=6), intra-arterial (n=7) and intramuscular groups (n=5). Plasmid DNA (phVEGF165) expressing vascular endothelial growth factor (VEGF) was obtained from HL60 cells, and transfection into CHO cells and western blot analysis of the medium, as well as proliferation assay of CPAE cells were performed. Two weeks after construction of the models, 500 mug phVEGF165 was injected into both the left common iliac artery and thigh muscles. Angiography was performed and the number of vessels counted, and ELISA was used to determine the quantity of VEGF in blood samples. Wilcoxon signed rank test was employed for statistical analysis. RESULTS: VEGF165 was expressed on western blot of the culture medium. Proliferation assay showed that optical densities were 0.73+/-0.043 in the control study and 1.09+/-0.015 in phVEGF165. The angiographic scores were 1.32+/-0.13 (pre-gene therapy) and 1.30+/-0.07 (post-gene therapy) in the control group, 1.42+/-0.15 and 1.59+/-0.09 in the intra-arterial group, 1.59+/-0.27 and 1.14+/-0.12 in the intramuscular group. The differences were not statistically significant. In the intra-arterial group, serum VEGF levels were 39.96+/-1.08 pg/ml (pregene therapy), 44.99+/-2.13 pg/ml (4th day), 48.18+/-1.49 pg/ml (1st week), 45.70+/-3.77 pg/ml (2nd week), and 46.54+/-5.47 pg/ml (3rd week), but in the control and intramuscular groups there were no increases. CONCLUSION: phVEGF165 affected the proliferation of CPAE cells. There was no difference in angiographic scores and serum VEGF levels between intra-arterial and intramuscular administrations.

Condensation mechanism of aminoglucomannan with plasmid DNA / 第三军医大学学报

Objective To study the condensation mechanism of aminoglucomannan(AGM)with plasmid DNA and some influencing factors in this process.Methods The polysaccharide-DNA complex was analyzed with agarose gel electrophoresis by observing the changes of DNA bands under various influencing conditions including different pH,ionic strength(NaCl concentration),temperature and plasmid DNA sorts.Results The new bands appeared in the slow migration positions after reaction between AGM and plasmid DNA,and the changes of DNA bands content were dependent on AGM concentrations.The condensation degree could be effected by pH,temperature,and ionic strength.Conclusion Our study suggests that the AGM can be condensed with plasmid DNA in proper conditions.

Enhanced Expressions and Histological Characteristics of Intravenously Administered Plasmid DNA in Rat Lung

Cationic liposome-mediated gene transfection is a promising method for gene therapy. In this study, the transfection efficiency and histological patterns were evaluated in rat lung after intravenous administration via femoral vein of naked plasmid DNA, naked plasmid DNA with pretreatment of DOTAP, and DOTAP-cholesterol-plasmid DNA complex. Plasmid DNA encoding bacterial LacZ gene was used. For quantification of LacZ gene expression, -galactosidase assay was performed. For histologic examination, X-gal staining and immunohistochemical staining for transfected gene products were performed. Pretreatment of DOTAP prior to the infusion of naked plasmid DNA increased transfection efficiency up to a level comparable to DOTAP-cholesterol-plasmid DNA complex injection. Transfected genes were mainly expressed in type II pneumocytes and alveolar macrophages in all animals. We conclude that the high transfection efficiency is achievable by intravenous administration of naked plasmid DNA with pretreatment of DOTAP, to a level comparable to DOTAP-cholesterol-plasmid DNA complex. In this regard, naked plasmid DNA administration with pretreatment of DOTAP could be a more feasible option for intravenous gene transfer than DOTAP-cholesterol-plasmid DNA complex, in that the former is technically easier and more cost-effective than the latter with a comparable efficacy, in terms of intravenous gene delivery to the lung.

An ELISA for detection of anti-dsDNA by using plasmid DNA as antigen / 中华检验医学杂志

Objective To develop an ELISA for detection of anti-dsDNA antibody by using plasmid DNA as antigen.Methods DNA in plasmid pBV220 for prokaryotic expression vector was purified by base-cleavage. The microtiterplates were pretreated by poly-L-lysine and coated by the plasmid DNA in a dilution of 1∶50 as antigen. An ELISA method for detection of serum anti-dsDNA antibodies was developed with HRP-SPA as enzyme-labeled marker.The IIF using crithidia lucilia as substrate was performed simultaneously for comparison. The serum samples from 64 patients with SLE, 8 with MCTD and 17 with RA were detected. Results The concentration of DNA was 1 54 g/L by UV spectrophotometer at wavelength of 260 nm. The positive percentage of ELISA for anti-dsDNA was higher than that of IIF. By comparison with IIF the positive percentages in SLE, MCTD and RA groups were 23 4% vs 17 2%, 12 5% vs 12 5% and 11 8% vs 5 9%, respectively, and the coincident rates between the 2 methods were 93 8%, 100% and 94 1% respectively. The sensitivity and specificity of the developed ELISA for detection of anti-dsDNA were 100% and 93 4% when IIF was as gold standard.Conclusion The ELISA by using plasmid DNA as antigen to detect anti-dsDNA has fine precision, sensitivity and specificity. Its positive rate is higher than that of IIF thus it will contribute to monitor the activities for SLE patients′ condition.

Comparison of serotypes, restriction enzyme analysis of plasmid DNA pattern and PFGE(pulsed-field gel electrophoresis) patterns of Yersinia pseudotuberculosis isolates in Korea / 대한임상병리학회지

BACKGROUND: Yersinia pseudotuberculosis, a member of genus Enterobactericeae, is a main etiologic organism of diarrhea in childhood. Because a mouse and a unchlorinated spring water are main reservoirs of Y. pseudotuberculosis, the strains from a contaminated spring water and mouse could be involved in human epidemic. The purpose of this study was to investigate a clonality between the strains from patients and those from an unchlorinated spring water and a mouse by restriction enzyme analysis of plasmid DNA and pulsed-field gel electrophoresis (PFGE). METHOD: We isolated 15 Y. pseudotuberculosis strains including 8 isolates from patients (S1-S8), 6 isolates from mountain water (W1-W6), 1 isolate from a mouse (M1) in northeast area of Seoul. Plasmid and chromosomal DNA of all strains were analyzed by REAP with Bam H1 restriction and by PFGE with Xba I restriction , respectively. RESULTS: Restriction enzyme analysis of plasmid DNA was classified into type B and type D. All 7 strains of serotype 15 were classified as type B and 8 strains of serotype 4b were classified as type D. PFGE were classified into 6 different types. Among them, strains of PFGE type I, II, III, IV belong to Y. pseudotuberculosis serotype 15 and Y. pseudotuberculosis 4b strains were classified into PFGE type V, VI. S1 and W1 were classified into PFGE type I . S8, W6 and M1 were classfied into PFGE type VI. CONCLUSIONS: PFGE revealed clonality among strains from patients, a water and a mouse. PFGE was more discriminative than REAP to characterize the Y. pseudotuberculosis outbreaks in Korea.