潮霉素B抗性为选择标记的整合型表达载体的构建及在米根霉中的遗传转化

为了建立适合米根霉的遗传转化体系,应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体p BS-hygro-ldh A;分别采用PEG/Ca Cl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体p BS-hygro-ldh A转化入米根霉AS 3.819菌株中,并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响;通过荧光定量PCR(q PCR)对米根霉转化子基因组中质粒整合拷贝数进行了检测,并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体p BS-hygro-ldh A的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高,原生质体电转化最佳电场强度为13 k V/cm,孢子萌发2.5 h转化率最高,萌发孢子电转化最佳电场强度为14 k V/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明,在一定范围内,高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系,为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。     

An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi.

A rapid and efficient electroporation procedure has been developed for transformation of germinating conidia of filamentous fungi. Pretreatment of conidial preparations with a cell wall weakening agent, such as beta-glucuronidase, was found to be essential for successful transformation. Using the qa-2+ gene of Neurospora crassa, encoding the catabolic dehydroquinase, as a selectable marker with a double-mutant host strain, auxotrophic for aromatic amino acids, integration of the plasmid was observed to be predominantly at ectopic chromosomal sites. Cotransformation with the qa-2+ gene and a plasmid containing a heat shock gene sequence (hsp70 of N. crassa) suggested integration site preference. High efficiencies of transformation to hygromycin resistance were achieved employing the bacterial hygromycin B phosphotransferase gene with N. crassa, the patulin-producer Penicillium urticae, and the causal agent of blackleg disease of crucifers, Leptosphaeria maculans. The economically important species Aspergillus oryzae was similarly transformed to benomyl resistance with the benomyl-resistant beta-tubulin gene of N. crassa as a dominant selectable marker.     

Genetic transformation of Lactobacillus casei by electroporation

Lactobacillus casei IAM1045 was transformed with a plasmid pAM beta 1-1, a tra deleted derivative of pAM beta 1, by electroporation. Effective transformation was achieved in electroporation buffers of a wide range of pH values, and in all phases of cell growth tested, with highest frequency in the early log phase. Polyethylene glycol increased the transformation frequency, whereas divalent cations such as Mg2+, Ca2+ and Mn2+ at 0.25 mM decreased the frequency by 2 to 3 orders. Highly efficient transformation of approximately 10(-4)/viable cell was achieved under optimal conditions. A plasmid harboring the trpD, C, F, B and A genes from L casei RNL7 was introduced by electroporation into tryptophan auxotrophic L casei JCM1053. The resulting transformant was found to express the trp genes introduced.     

The particle inflow gun can be used to co-transform Paramecium using Tungsten particles

A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin-resistance gene, obtaining a transformation efficiency of 0.31+/-0.14% (mean+/-SD) for tungsten particles and 1.30+/-0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71+/-20%) a Paramecium mutant strain could be phenotypically rescued by co-transformation with a second plasmid containing the selectable neomycin-resistance gene. The PIG coupled with tungsten particles as the carrier offers a low-cost alternative for biolistic transformation of Paramecium.     

Strong extracellular nuclease activity displayed by barley (Hordeum vulgare L.) uninucleate microspores

 Electroporation is becoming an increasingly important technique for plant transformation. Nevertheless, no positive results were achieved in barley when uninucleate microspores were used as target cells. Since it was previously demonstrated that electric shocks create pores in the microspore cell wall, experiments were designed to verify the presence of nucleases in the electroporation mix. Aliquots of all the solutions used for microspore extraction, purification and transformation were collected and analysed using supercoiled pBI 221 as a substrate; a nuclease activity was detected in all samples. Though microspore rinsing removed most nucleolytic activity in the supernatants, DNA preservation in the electroporation buffer was difficult to achieve, because microspores appeared capable of synthesising and releasing endonucleases at any time. Microspore chilling at 0°C was fairly effective in reducing nuclease secretion in the mix, whereas 1%PEG or 10 mM EDTA maintained most of the DNA in a supercoiled or circular relaxed form. EDTA effects were counterbalanced by Mg²⁺, but not Ca²⁺ or Zn²⁺, and enhanced by Mn²⁺. Barley microspore nucleases actively degraded different DNAs as well as TMV RNA, and apparently had a molecular weight above 30 kDa. Nuclease inactivation with EDTA did not alter microspore viability and allowed a transient expression of the uidA gene in electroporated barley microspores. Received: 13 January 1997 / Accepted: 28 February 1997     

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.     

Study of the structure of metal-binding centers of calmodulin by EXAFS-spectroscopy

An investigation of Ca2+-binding centers of calmodulin was carried out by EXAFS-spectroscopy. The experimental results for protein preparations of calmodulin in which Ca2+ was isomorphically replaced by Tb3+ were obtained by a spectrometer working at the Institute of Nuclear Physics. For spectra analyses a standard method of Fourier transformation was used. Coincidence main maxima on phi (r) curves and identity of Fourier transformation for calmodulin and parvalbumin in the 2-6 A interval allow to infer the identity of Ca2+-binding centers of calmodulin and parvalbumin.     

DNA transfection of Escherichia coli by electroporation

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.     

Electroporation of rapeseed protoplasts – transient and stable transformation

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10⁻⁵ and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.     

The effect of cell synchronization on the efficiency of stable gene transfer by electroporation

We synchronized thymidine kinase deficient mouse Ltk-cells by two different methods, hydroxyurea double-block treatment or aphidicolin single-block treatment and transformed them with the cloned herpes simplex virus thymidine kinase gene at various time intervals by the electroporation technique. Marked enhancement of stable transformation efficiency was observed at the time corresponding to the peak of G2/M phase. These results suggest that the G2/M phase is the most efficient period for stable gene transfer by electroporation.     

Transfer of MHC genes into hematopoietic stem cells by electroporation: A model for monitoring gene expression

Abstract

Previous attempts in using recombinant viral vectors to transfer genes into bone marrow cells have resulted in expression frequencies of 2‐15% (8,9,12,13,14,24). These vectors, however, require complex gene vector constructions and culture methods and have been difficult to utilize. We therefore have used electroporation to establish a less complicated and equally efficient technique for gene transfer. Conditions yielding high gene transfer into bone marrow precursor cells by electroporation were determined using a mutant dihydrofolate reductase gene and/or murine MHC class II genes. Successful transfer and expression of these genes were assessed by (1) fluorescence staining using monoclonal antibodies, (2) acquired resistance to the anti‐folate drug methotrexate and (3) Southern blot analysis. Stable integration of the newly acquired mutant dihydrofolate reductase gene was observed in > 25% of murine hematopoietic progenitor cells (CFU‐M). Electroporation of class II genes in conjunction with the mutant dihydrofolate reductase and subsequent selection with methotrexate resulted in expression of transfected la molecules in 12‐15% of bone marrow derived macrophages. Integration of the transferred major histocompatibility genes was verified by Southern blot analysis of high molecular weight cellular DNA following electroporation and methotrexate selection. The frequencies of gene transfer and expression achieved in this study suggest that electroporation is a viable option for monitoring gene expression in bone marrow derived cells.     

Generation of a conditional allele of the mouse prostaglandin EP4 receptor

Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus areteriosus (PDA). To circumvent this, an EP4 allele amenable to conditional deletion using the Cre/loxP system was generated. The targeting construct was comprised of a floxed exon2 in tandem with the neomycin-resistance gene in intron 2, flanked by third 3' LoxP site. Mice homozygous for the targeted allele (EP4(lox+neo/lox+neo)), or following its Cre-mediated deletion (EP4(del/del)), also die within hours of birth with PDA. In contrast, mice homozygous for a partially recombined allele, retaining exon2 but lacking neo (EP4(flox/flox)), are viable and show no overt phenotype. Postnatal deletion of the floxed EP4 gene is efficiently achieved in the liver and kidney in a transgenic mouse expressing the inducible Mx1Cre recombinase. The EP4(flox) mouse should provide a useful reagent with which to examine the physiologic roles of the EP4 receptor.     

Transformation of cauliflower (Brassica oleracea L. var. botrytis) — an experimental survey

The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.     

Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 × 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 × 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.     

Cloning of the<Emphasis Type="Italic"> KcURA3</Emphasis> gene and development of a transformation system for<Emphasis Type="Italic"> Kluyveromyces cicerisporus</Emphasis>

KcURA3 was cloned from Kluyveromyces cicerisporus CBS4857 by complementation of the ura3 mutation in Saccharomyces cerevisiae. KcURA3 encodes a 267-amino-acid protein with 80% sequence identity to that of S. cerevisiae. An ura3 mutant strain from K. cicerisporus CBS4857, named Y179U, was obtained by selection on 5-fluoroorotic acid plates. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +277. Two vectors, pUK1 and pUKD, bearing KcURA3 were constructed. Either the lithium acetate method or electroporation could be used to transform pUK1 and pUKD intoY179U. The transformation efficiency using electroporation was higher than that using the lithium acetate method.     

Transformation of Bacillus thuringiensis by electroporation

Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 10(5) transformants/micrograms. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 10(2) pC194+ pUB110 transformants/micrograms DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and in acrystalliferous mutant of the same strain transformed at frequencies of 10(4)-10(5)/micrograms DNA with most of the plasmids tested. A cloned israelensis 27-kDa delta-endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.     

The effective method of gene transfer into mammalian cells cultured in vitro

Efficiency of transformation by a number of vectors with the different selective markers of a set of cell lines has been studied for three different methods based on using calcium phosphate, polybrene, or electroporation. Electroporation is shown to be the most efficient one. Using this method with the system rat2k-cells-pAGO vector we have obtained the frequencies of transformation up to 2-3.10(-3). We suggest to use this system as a model for investigation of homologous recombination in the framework of the gene therapy project.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.