Improved intracellular delivery of oligonucleotides by square wave electroporation

Prior studies have shown that electroporation is a simple and effective method for the introduction of oligonucleotides (ODN) into cells. In ex vivo bone marrow purging models, electroporation of ODN into cells has been associated with selective killing of human neoplastic cells while sparing hematopoietic stem cells. Prior studies used conventional electroporation methods (i.e., exponential decay) to introduce ODN into cells. Square wave electroporation allows the delivery of a more defined and regulated electrical pulse and is associated with high transfection efficiencies in a variety of systems. The current study was undertaken to determine whether square wave electroporation was more effective than exponential decay electroporation for the delivery of ODN into hematopoietic cells. Using fluorescein-tagged ODN and K562, chronic myelogenous leukemia (CML) cells, higher transfection rates were observed after square wave electroporation. In addition, c-myc antisense ODN were more effective in reducing c-myc protein when introduced by square wave electroporation, as compared with introduction by exponential decay electroporation. Square wave electroporation is thus identified as the optimal method for delivering ODN into hematopoietic cells.     

Desalted deep sea water increases transformation and homologous recombination efficiencies in Dictyostelium discoideum

The life cycle of Dictyostelium discoideum consists of many cellular and developmental aspects. By virtue of its relatively high transformation efficiency and a small haploid genome, this organism has proven to be advantageous for characterizing gene functions. However, a much higher transformation efficiency is required as one of the prerequisites for unraveling gene function on a genome-wide scale. In this study, we describe the positive effect of desalted deep sea water, when used as a solvent medium, on the transformation and homologous recombination efficiencies in Dictyostelium. A standard Dictyostelium medium HL5 containing desalted deep sea water, HL5dsw, distinctly increased both the transformation and homologous recombination efficiencies by approximately 2- to 3-fold. Furthermore, we observed that the growth of cells in HL5dsw both before and after electroporation contributed to the increase in transformation efficiency. These results indicate that a simple modification of the solvent medium remarkably enhanced the isolation of transformants and gene-targeted clones, which had previously been difficult to isolate.     

High efficiency transformation of E. coli by high voltage electroporation.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.     

Transformation of Dictyostelium discoideum with plasmid DNA

DNA-mediated transformation is one of the most widely used techniques to study gene function. The eukaryote Dictyostelium discoideum is amenable to numerous genetic manipulations that require insertion of foreign DNA into cells. Here we describe two commonly used methods to transform Dictyostelium cells: calcium phosphate precipitation, resulting in high copy number transformants; and electroporation, an effective technique for producing single integration events into genomic DNA. Single integrations are required for gene disruption by homologous recombination. We also discuss how different selection markers affect vector copy number in transformants and explain why blasticidin has become the preferred selectable marker for making gene knockouts. Both procedures can be accomplished in less than 2 h of hands-on time; however, the calcium phosphate precipitation method contains several incubations, including one of at least 4 h, so the total time required for the transformation is approximately 8 h.     

小麦转基因方法及其评述

小麦是遗传转化比较困难的作物之一。为了克服小麦基因工程育种和功能基因组学研究的障碍,人们分别尝试利用基因枪、花粉管通道、超声波、离子束注入、激光微束穿刺、PEG(Polyethylene glycol)、电击和农杆菌等方法转化小麦,涉及的受体材料包括幼胚、成熟胚、花药愈伤组织、幼穗、芽尖和花器官。文章对小麦主要遗传转化方法及其应用进行了介绍、回顾和评述,分析、比较了获得安全型转基因小麦的几种策略,以期增强读者对小麦转基因技术和进展的了解,促进小麦转化技术的持续改进和提高。     

Establishment of the nuclear location of Dictyostelium discoideum plasmids

The nuclear location of the Dictyostelium discoideum plasmids was studied using a biochemical approach based on the presence of plasmid sequences in nucleosomes. This analysis revealed that all four of the known plasmids (Ddp1, Ddp2, Ddp3, Ddp5) are present in chromatin. This evidence establishes that the D. discoideum plasmids are not cytoplasmic but located in the nucleus. D. discoideum is unique among eukaryotes in possessing a group of nonhomologous endogenous plasmids in its nucleus. These plasmids are excellent starting material for construction of nuclear transformation and expression vectors. Such vectors upon transformation into D. discoideum are also present in chromatin as expected for DNA located in the nucleus.     

A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substrates

Electroporation is an important approach for genetic engineering experiments allowing for introduction of foreign DNA in a selected host. Here, we describe for the first time the use of glycine betaine as an osmoprotectant for electroporation of gram-positive bacteria Bacillus subtilis. High electroporation efficiency (up to 5×10(5) cfu/μg) was obtained using 7.5% glycine betaine. The new method improved the transformation efficiency of B. subtilis with linear integrative DNA nearly 700-fold compared with existing Bacillus transformation techniques.     

红曲霉不同转化方法比较

为了研究红曲霉聚酮体途径,考察和比较了4种不同的转化方法以建立有效的红曲霉遗传转化系统。以潮霉素作为抗性筛选标记,pBC-Hygro作为转化载体,用基于原生质体的传统转化和电击转化、基于萌发孢子的电击转化以及REMI技术转化红曲霉。发现基于萌发孢子的电击转化由于转化率极低而不适于红曲霉转化。基于原生质体的传统转化和电击转化尽管每微克DNA分别能获得135个转化子和125个转化子,但因转化子稳定性差也适合红曲霉转化的转化。应用REMI技术,转化率提高约20倍,每微克DNA 2500个转化子,70%～75%的转化子的稳定,非常适合于红曲霉的转化。      

Analysis of proportion regulation in slugs of Dictyostelium discoideum using a monoclonal antibody and a FACS-IV

A quantitative assay for estimating the proportion of prespore cells in D. discoideum slugs was established by labelling disaggregated slug cells with a prespore specific monoclonal antibody and analysing the cell population with a FACS-IV. The method is validated using a wild-type strain and its stalky mutant. "Wild-type" strains have different proportions of prespore cells and it is demonstrated that slugs of some strains have an increased percentage of prespore cells when migrated in the dark compared to the light and in the presence of EGTA. The technique is rapid and will make possible genetic analysis of proportion regulation in D. discoideum.     

Isolation and characterization of a mildly thermophilic nonsulfur purple bacterium containing bacteriochlorophyll b

A method for transformation of whole Bacillus amyloliquefaciens cells by electroporation was developed. The procedure is as efficient as the protoplast transformation method, resulting in up to 10(5) transformants/micrograms plasmid DNA, but requires less effort and time. Cells for electroporation were grown to late exponential phase in a rich medium supplemented with 0.25 M sucrose, washed with and resuspended in 0.25 M sucrose, 1 mM HEPES, 1 mM MgCl2, 10% (v/v) glycerol, pH 7.0, at 3-5 x 10(10) cells/ml for storage at -80 degrees C. The highest transformation frequency was obtained at 7.5 kV/cm with a 25 microF capacitor. The transformation efficiency increased linearly with DNA concentration at least over the range 10 ng-12.5 micrograms/ml. Transformations with ligated DNA and of industrial strains were also successful. In addition, B. subtilis cells treated as above could be transformed by electroporation, resulting in 10(4) transformants/micrograms DNA at 12.5 kV/cm.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.     

Optimized transformation by electroporation ofLactobacillus plantarum strains with plasmid vectors

Summary Whole cell transformation ofLactobacillus plantarum CCM 1904 by electroporation was optimized. Pulse duration and electric field strength were shown to be important parameters: the optimum conditions were 12.5 kV/cm, a time-constant of 10 ms for an exponential decay waveform and 6.7 kV/cm applied during 2.5 ms for a square waveform. Transformation efficiency was increased if cells were cultivated on medium containing sorbitol and harvested during their early exponential growth phase: 8 × 10^–4 transformants/g pGK12 DNA per viable cell were obtained, with a survival rate of 10%–30% Cryotreatment by several freeze-and-thaw cycles decreased transformant yields. Transformation efficiency with different plasmids was studied and plasmid pGK12 was found to transformL. plantarum the most efficiently. Transformation by electroporation ofL. plantarum is strain dependent. The best results were obtained withL. plantarum NCIB 7220, giving 5 × 10⁶ transformants/gmg plasmid pGK12 DNA.     

Quantification of DNA uptake by Dictyostelium discoideum amoebae and the stability of the DNA during growth and development

We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

How does environment modify the spatial structure of Dictyostelium discoideum population

The transformation of the spatial structure of a Dictyostelium discoideum population in response to environmental changes induced by this population was investigated. A comparative analysis of the spatial and temporal characteristics of the D. discoideum colony is given for two cases: (a) when the colony is cultivated on a bacterial lawn, i.e. under conditions close to natural, and (b) in the absence of the bacterial lawn when the colony grows on the nutrient substrate enriched with folic acid. It is shown that the environmental changes induced by cell metabolism modify the spatial structure of the D. discoideum population first, the rate of population propagation falls drastically, which correlates with a decrease in the substrate pH; second, the spatial redistribution of the D. discoideum cell density correlates with the redistribution of folic acid in the substrate. The mechanism of the environment impact on the D. discoideum colony transformation is discussed.     

Effects of pulse length and pulse strength on transfection by electroporation

The relative importance of pulse field strength E and pulse length tau 1/2 (half decay time of an exponential decay pulse) on the stable transfection frequency for HeLa or HUT-78 cells was investigated. Cells were transfected with plasmids containing the promoter and drug resistant genes pRSVgpt or pRSVneo by electroporation. The stable transfection frequency was assayed using the marker rescue technique. The transfection frequency increases with increasing values of E tau 1/2. For a given pulse length, the transfection frequency is proportional to the power of the pulse (E2 tau 1/2). Pulses with half decay times of 2.2 to 4.6 ms appear to be more efficient than 0.275 to 0.31 ms for stable transfection of HeLa cells.     

Efficient transformation ofKlebsiella oxytoca by electroporation

A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a high transformation efficiency. The highest efficiency of 1.6 × 10⁶ transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD₆₀₀ of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF.     

Optimization of electroporation conditions for Arthrobacter with plasmid PART2

A prerequisite for genetic studies of Arthrobacter is a high efficiency transformation system that allows for DNA transfer, transposon mutagenesis, and expression of specific genes. In this study, we develop a detailed electroporation method through a systematic examination of the factors involved in the entire electroporation process. Key features of this procedure, including the addition of penicillin to cells during the early log phase of growth and the presence of 0.5 M sorbitol in the electroporation and recovery media, produced the greatest increases in transformation efficiency and consistency of results. The transformation rate also varied depending on the electrical parameters, DNA concentration, and recovery time period. Using optimum conditions, we generally achieved an efficiency of 6.8 × 10⁷ transformants per microgram of PART2 for Arthrobacter sp. A3. This protocol was also successfully applied to other Arthrobacter species. Therefore, we conclude that the proposed method is rapid, simple and convenient, which allows a transformation trial to be accomplished in minutes.     

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10⁵/μg of DNA to 10⁷/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.