Microgram Of Plasmid DNA Research Articles

Duplication and Transformation of the Schizosaccharomyces pombe Collection of Deletion Strains.

We present an efficient and organized method of lithium acetate and polyethylene glycol-based transformation of plasmid DNA into the commercially available collection of Schizosaccharomyces pombe with single-gene deletions. We also describe how to prepare a duplicate collection of the deletion strains in order to preserve the longevity of the master set. These protocols are adapted to the 96-well format of the 3004 strains of the Version 2.0 Bioneer set but can also be used for later releases of the collection. This transformation method typically yields efficiencies in the range between 1.0×103 and 1.0×104 transformants per microgram of plasmid DNA. However, some deletion strains transformed with significantly lower efficiencies. We provide a list of these difficult-to-transform strains. Applications for this methodology include the transformation of the deletion set with plasmids necessary for genetic screens.

Genetic transformation of moss Ceratodon purpureus by means of polycationic carriers of DNA

There is much progress in application of genetic engineering for improving the biological properties of different organisms. Viral and nonviral carriers are used for delivery of genetic material into target cells. Polymeric materials of natural and synthetic origin are the most promising gene delivery agents. These polymers demonstrated high efficiency of DNA delivery into animal cells, although they were not very effective in plant cells. Here, the procedure for genetic transformation of Ceratodon purpureus (Hedw.) Brid. moss protoplasts is described. The method is based on the application of surface-active polymeric carriers of the poly-DMAEM structure and controlled length and charge. This allows obtaining more transient and stable moss transformants per microgram of plasmid DNA when compared with known protocol based on using polyethyleneglycol. It is easier, more convenient, and cheaper than the “gene gun” method. Prospects for further improvement of structure and functional characteristics of new polymeric carriers are considered for delivery of genetic material into plant cells.

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10(8) and 10(6) transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

New method for electroporation of Lactobacillus species grown in high salt

We here describe a new method for electroporation of Lactobacillus species, obligately homofermentative and facultatively heterofermentative, based on the cell-wall weakening resulting from growth in high-salt media. For L. casei, optimum transformation efficiency of up to 10 5 transformants per microgram of plasmid DNA was achieved following growth in the presence of 0.9 M NaCl. Plasmids of different sizes and replication origins were also similarly transformed. These competent cells could be used either directly or stored frozen, up to 1 month, for future use, with similar efficiency. This protocol was assayed with different Lactobacillus species: L. delbrueckii subsp. lactis, L. paracasei, L. plantarum and L. acidophilus, and it was found that they were transformed with similar efficiency.

Expression of alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae in Ashbya gossypii

Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 alpha (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10-20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg-1 of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l-1, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.

165. Long-Term Factor VIII Expression and Phenotypic Correction in a Murine Model of Hemophilia A Achieved by Immunosuppression and Non-Viral Gene Transfer Using the Sleeping Beauty Transposon

Hemophilia A is an X-linked recessive genetic disorder that is characterized by sustained bleeding after trauma or injury. Currently, patients are treated by self-administration of recombinant factor VIII protein (FVIII), but this treatment is expensive and cumbersome. Gene therapy using viral vectors has been successfully used to correct the bleeding disorder in FVIII deficient mice and dogs. However, anti-viral immune responses, problems with scale-up, and insertional mutagenesis, due to a predilection for most integrating viruses to insert near genes, all raise safety and practicality questions for viral vectors. Therefore we used the Sleeping Beauty (SB) transposon system, which is delivered as plasmid DNA, to integrate a B-domain deleted FVIII genes into the chromosomes of C57BL/6J-F8−/− mice and correct the phenotype. The transposon plasmid was co-delivered with a Ubiquitin 3 promoter-regulated SB 10 transposase expression plasmid using the “hydrodynamic” method. Plasma FVIII levels were assayed in treated mice by ELISA, COAT test, and APTT clotting assay. Long-term FVIII expression (>9 weeks at present) required delivery of 280 micrograms of plasmid DNA, co-delivery of Ub-SB10 plasmid, and immunosuppression by bi-monthly 150 mg/kg cyclophosphamide injections or immunotolerization by FVIII (RefactoTM) injection in C57BL/6J-F8−/− neonates. Without immunosupression or tolerization, anti-FVIII antibody was detected in treated mice using the Bethesda assay. Transposon excision products were detected in DNA extracted from the liver of mice treated with transposon and transposase, providing evidence for transposition. Transposon insertion site analyses in hepatocytes from treated mice are ongoing. The SB transposon system thus provides non-viral means for obtaining stable FVIII gene insertion and expression for the treatment of hemophilia A.

Homologous expression of Phanerochaete chrysosporium manganese peroxidase, using bialaphos resistance as a dominant selectable marker.

Manganese peroxidase (MnP) is a major extracellular component of the lignin-degrading system of the white-rot fungus, Phanerochaete chrysosporium. Homologous expression of recombinant MnP isozyme 1 (rMnP1) in P. chrysosporium was achieved using a novel transformation system for this fungus, which utilizes the Streptomyces hygroscopicus bialaphos-resistant gene, bar, as the selectable marker. The transformation frequency for this system is approximately 100 bialaphos-resistant transformants per microgram of plasmid DNA. Transformed strains all contain plasmid DNA, ectopically integrated into the fungal genome. Using this transformation system, the promoter region of the P. chrysosporium translation elongation factor gene was used to drive expression of mnp1, encoding MnP1, in primary metabolic cultures of P. chrysosporium, where endogenous MnP was not expressed. Approximately 2-3 mg of active recombinant MnP1 per liter of extracellular medium was produced in agitated cultures of transformants.

Vascular endothelial growth factor (VEGF) fails to improve blood flow and to promote collateralization in a diabetic mouse ischemic hindlimb model

BackgroundAngiogenic therapy with vascular endothelial growth factor (VEGF) has been proposed as a treatment paradigm for patients suffering from an insufficiency of collateral vessels. Diabetes is associated with increase in the production of VEGF and therefore additional VEGF may not be beneficial. Accordingly, we sought to determine the efficacy of VEGF therapy to augment collateral formation and tissue perfusion in a diabetic mouse ischemic hindlimb model.MethodsDiabetic and non-diabetic mice were studied in parallel for the efficacy of VEGF administration. Diabetes was induced with streptozotocin. Hindlimb ischemia was produced by severing the left iliac artery. An outlet tube from an osmotic infusion pump with placebo/ 500 micrograms of plasmid-DNA encoding VEGF was fenestrated and tunneled into the left quadriceps muscle.ResultsVEGF induced more rapid and complete restoration of blood flow in normal mice. However, in the setting of diabetes there was no difference between VEGF Vs. placebo in the rate or adequacy of flow restoration. There was a significant increase in smooth muscle actin and Factor-VIII antigen densities in diabetic animals and in animals which received VEGF.ConclusionsAngiogenic therapy with VEGF in the setting of diabetes does not appear to have the beneficial effects seen in the absence of diabetes.

Electrotransformation studies in Clostridium cellulolyticum

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV x cm(-1) field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV x cm(-1) pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10(5) and 10(7) transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively.

CBF/NF-Y functions both in nucleosomal disruption and transcription activation of the chromatin-assembled topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in chromatin is dependent on the promoter structure.

To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.

A set of Hansenula polymorpha integrative vectors to construct lacZ fusions

A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around 100 transformants per microgram of plasmid DNA; in all transformants checked by Southern blotting the plasmid was integrated into the genome locus corresponding to the gene plasmid marker. PCR showed that about 50% of the transformants contained more than one plasmid copy per genome. Experiments carried out using the developed plasmids to determine the strength of the gene promoters involved in nitrate assimilation in H. polymorpha revealed that, in the presence of nitrate, the nitrate reductase gene promoter (YNR1) was the strongest, followed by nitrite reductase (YNI1) and nitrate transporter (YNT1).

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Transformation of a flocculating Saccharomyces cerevisiae using lithium acetate and pYAC4

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 103 transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one.

Transformation of a flocculatingSaccharomyces cerevisiae using lithium acetate and pYAC4

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 10(3) transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one.

Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance.

Germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved using lithium acetate treatment or electroporation. The efficiency of transformation was up to 32 and 25 transformants per microgram of plasmid DNA with the two methods, respectively. Transformants obtained by the lithium acetate method were more stable and showed a high copy number of the hph gene integrated into their genome. The other transformants, from the electroporation procedure, were stable, but unable to grow in the presence of high levels of hygromycin, and detection of the hph gene was only possible by polymerase chain reaction analysis.

Human gene therapy for melanoma: CT-guided interstitial injection.

Our intent is to describe the role of CT in the intratumoral injection of Allovectin-7 (Vical, San Diego, CA), an allogeneic class I major histocompatibility complex antigen, HLA-B7, formulated with cationic lipid, in the treatment of metastatic malignant melanoma. Ten patients with metastatic malignant melanoma were treated with gene therapy in which we used CT-guided intratumoral injection of plasmid DNA containing the HLA-B7 gene. This therapy was part of a phase I gene therapy trial in patients with metastatic melanoma. CT guidance was chosen as an accurate way to direct gene delivery in patients with deep, impalpable lesions. Tumor locations included pulmonary, mediastinal, hepatic, adrenal, and paracaval sites. Patients in the CT protocol underwent baseline CT studies. Examinations were repeated 2, 4, and 8 weeks after gene therapy and thereafter at 3-month intervals. Both injected and noninjected tumors were measured. CT-guided injections of 10, 50, or 250 micrograms of plasmid DNA were performed with 22-gauge spinal needles. Injection volumes were between 1.0 and 4.0 ml, depending on tumor size. CT-guided core biopsy specimens were obtained (with 18- or 20-gauge needles) from the selected tumor before therapy and 2, 4, and 8 weeks after therapy to assess HLA-B7 plasmid DNA and gene expression. Peripheral blood was analyzed for cytotoxic T lymphocytes directed against HLA-B7. CT-guided intratumoral injections were successful in delivering genetic material to all patients with impalpable tumors. Significant responses (as defined by a decrease of 25% or more in the product of the length and width of the injected tumor) were observed in six of the 10 patients. One of these six patients who had a solitary lesion remains free of disease 19 months after gene therapy. HLA-B7 protein expression was detected in 89% of biopsy specimens, and plasmid DNA and messenger RNA were detected in 56% and 22% of biopsy specimens, respectively. CT provides a safe, accurate, and efficacious way to monitor and assess tumor progression and response, and it provides guidance for biopsies and intratumoral injections during gene therapy. Significant responses in injected tumors of six of the 10 patients in our study suggest that further clinical trials of this gene therapy are warranted.

Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration.

The factors controlling the transfection efficiency of cationic lipid carrier systems following intravenous administration are poorly understood. Using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) combined with Tween 80 as a carrier system and cDNA of luciferase or beta-galactosidase gene as a reporter, we investigated the importance of DOTMA to DNA ratio and the ratio of DOTMA to Tween 80 in the lipid formulation in determining the site and level of transgene expression following intravenous administration. The data show that all of the internal organs, including lung, liver, spleen, heart and kidneys, expressed the transgene upon systemic administration into animals with 25 micrograms of plasmid DNA when complexed with DOTMA-Tween 80 lipid formulation. The transfection efficiency was dependent on both DOTMA to DNA, and DOTMA to Tween 80 ratios. Among the organs examined, the lung appeared to be more transfectable than other organs. A better transfection activity was obtained with higher DOTMA to DNA and DOTMA to Tween 80 ratios. Time-response curve shows that gene expression was transient with a maximal level between 10 and 24 h after injection. Results from tissue distribution studies with 125I-labeled plasmid DNA and Southern analysis suggest that the transient expression is the result of the loss of transgene from the transfected cells. These results suggest that cationic lipid-based delivery systems can be efficient for gene delivery if the composition of the DNA-lipid complexes is properly controlled.

Transformation ofBurkholderia pseudomalleiby Electroporation

Conditions were defined for the successful transformation of the human pathogenBurkholderia pseudomallei4845 by electroporation, using the incQ plasmid pKT230. We have obtained frequencies of up to 8 × 103transformants per microgram plasmid DNA with freshly prepared electrocompetentB. pseudomallei.The method also allows for easy and reproducible production of frozen cell suspensions which can produce efficiencies up to 2.5 × 102transformants per microgram of plasmid DNA. Kanamycin had to be added to the culture growth medium at a minimum concentration of 20 μg ml−1and a maximum concentration of 50 μg ml−1for the bacteria to become electrocompetent. Bacteria grown in medium containing a final concentration of 30 μg ml−1kanamycin produced the highest numbers of transformants, although the transformation frequency at this concentration was not as efficient as that at 50 μg ml−1. Electron microscopy indicated that the kanamycin affects the integrity of the bacterial outer membrane, which becomes loose and wrinkled in appearance. The electrocompetence does not result in a permanent morphological change.

Transformation of the Methylotrophic ActinomyceteAmycolatopis methanolicawith Plasmid DNA: Stimulatory Effect of a pMEA300-Encoded Gene

Amycolatopsis methanolicacontains a 13.29-kb plasmid (pMEA300) present both in the free state and integrated at a unique genomic location. A pMEA300-free derivative (strain WV1) was selected, allowing further analysis of pMEA300-encoded functions. Whole cells of strain WV1 could be transformed at high frequencies (approximately 106transformants per microgram of plasmid DNA) with both circular and linear plasmid DNA, provided that the pMEA300-encodedstf(stimulation of transformation frequency) gene was present.stfwould encode a putative protein of 373 amino acids withMr40,201, resembling putative regulatory proteins involved in sporulation ofStreptomyces griseusandStreptomyces coelicolor.