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.     

In vivo electroporation and ubiquitin promoter--a protocol for sustained gene expression in the lung

BACKGROUND: Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. METHODS: The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 microl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. RESULTS: Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932+/-249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382+/-318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989+/-710 RLU/mg of total lung protein) with a peak at day 20 (2821+/-2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. CONCLUSIONS: These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved.     

An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.     

Electrotransfer in differentiated myotubes: a novel,efficient procedure for functional gene transfer

Development of reliable techniques for experimental manipulation of gene expression in multinucleated skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and pathophysiology. At present, viral vectors represent the only method to obtain efficient gene transfer in terminally differentiated myotubes. Here we present an in vitro procedure that relies on the application of a pulsed electric field for transferring naked DNA into differentiated myotubes seeded on coverslips. Compared with standard transfection methods, electroporation was at least 1000 times more efficient, as judged by quantitative determination of luciferase content. Percentage of transfected myotubes averaged around 45%. Moreover, we were successful in transfecting a dominant-negative ADP ribosylation factor 1 (ARF1) mutant, i.e., ARF1N126I, in myotubes, thus interfering with endoplasmic reticulum-Golgi traffic, as indicated by alterations of subcellular distribution of GM130, a cis/medial-Golgi marker. Co-transfection experiments with beta-galactosidase also showed that the ARF1 mutant appeared to inhibit myoblast fusion and could not be used before myotube formation. The present work validates the use of electroporation as a highly efficient approach for gene transfer in fully differentiated myotubes.     

Efficient transformation of Dictyostelium discoideum with a particle inflow gun

We report experiments to transform Dictyostelium discoideum using a simple home-made particle gun. Stable transformants were obtained at frequencies of up to 2500 clones/microg DNA. This is five times more than we achieve with the same vector using electroporation protocols. We also show that the particle inflow gun can be used for analysis of developmentally regulated gene expression in a transient assay.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Cre-<Emphasis Type="Italic">loxP</Emphasis> recombination-based reporter system for plant transcriptional expression studies*

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, beta-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.     

Cloning Vectors for Rice

We developed various binary vectors that can be used for expressing a foreign gene in rice. Vectors pGA3426, pGA3436, and pGA3626 are intended for overexpression of a gene using the maize Ubiquitin promoter, whereas pGA3780 is for rather mild expression of a gene using the rice Actin1 promoter. Vector pGA3777 is for expressing two genes simultaneously. We also developed binary vectors for expressing a fusion protein with a tag. Four vectors (pGA3427, pGA3428, pGA3429, and pGA3438) are for protein tags with sGFP, HA, His, and Myc, respectively. Vector pGA3383 is for analyzing promoter activity using the GUS reporter. In this vector, multiple cloning sites in front of GUS can be utilized for accepting a promoter fragment. We also generated transient expression vectors for studying the subcellular localization of a protein. Vectors pGA3452, pGA3651, and pGA3652 are for GFP fusion; pGA3574 for RFP fusion; pGA3697 for Myc tag; and pGA3698 for HA tag. In addition, we generated pGA3506, pGA3516, pGA3592, and pGA3593 for facilitating the subcloning of full-length cDNA clones into our binary vectors.     

Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer

Summary The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.     

An Adenoviral Vector-Based System to Study Neuronal Gene Expression: Analysis of the Rat Tyrosine Hydroxylase Promoter in Cultured Neurons

Abstract: We validated an adenoviral vector-based system as a move toward the characterization of regulatory sequences that are involved in the control of cell-type specificity and ligand regulation of neuronal gene expression in cultured neurons. We constructed recombinant adenoviruses, incorporating the luciferase gene under the control of different fragments of the rat tyrosine hydroxylase (TH) promoter. Similar results for luciferase expression were obtained in immortalized cells either by infection using adenoviral constructs or by transfection using conventional plasmid vectors. Taking advantage of adenoviral vectors, we extended our experiments to various primary cell cultures. The first 800 bp of the TH promoter were found to be sufficient to confer a cell-type preferential activity in noradrenergic neurons of the rat superior cervical ganglia. Furthermore, using this neuronal culture model, we showed that the same promoter region carries leukemia-inhibitory factor (LIF)-responsive element(s). Our results demonstrate that the first 800 bp of the rat TH promoter contains a functionally important core region for constitutive and LIF-regulated expression of TH in peripheral noradrenergic neurons. Moreover, the study validates the adenoviral vector-based system as a new strategy for studying the regulation of neuronal gene expression.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.     

A series of Dictyostelium expression vectors for recombination cloning

We describe a series of Dictyostelium expression vectors for recombination cloning using the Gateway technology. DNA fragments generated by high fidelity polymerase chain reaction are cloned by topoisomerase-mediated ligation, then recombined into any of several Dictyostelium expression vectors using phage lambda LR recombinase. No restriction enzymes are used in this procedure. Coding regions can be expressed from their own promoters, or from a strong actin 15 promoter as a native protein, or with an amino or carboxyl-terminal GFP fusion. Gene promoters of interest can be analyzed by controlled expression of GFP and beta-galactosidase. These vectors allow for rapid and simple characterization of novel DNA, and are ideal for high-throughput studies.     

A Series of TA-Based and Zero-Background Vectors for Plant Functional Genomics

With the sequencing of genomes from many organisms now complete and the development of high-throughput sequencing, life science research has entered the functional post-genome era. Therefore, deciphering the function of genes and how they interact is in greater demand. To study an unknown gene, the basic methods are either overexpression or gene knockout by creating transgenic plants, and gene construction is usually the first step. Although traditional cloning techniques using restriction enzymes or a site-specific recombination system (Gateway or Clontech cloning technology) are highly useful for efficiently transferring DNA fragments into destination plasmids, the process is time consuming and expensive. To facilitate the procedure of gene construction, we designed a TA-based cloning system in which only one step was needed to subclone a DNA fragment into vectors. Such a cloning system was developed from the pGreen binary vector, which has a minimal size and facilitates construction manipulation, combined with the negative selection marker gene ccdB, which has the advantages of eliminating the self-ligation background and directly enabling high-efficiency TA cloning technology. We previously developed a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, subcellular localization analysis and promoter activity detection. Our results show that such a system is highly efficient and serves as a high-throughput platform for transient or stable transformation in plants for functional genome research.     

萤光素酶表达质粒pEE14.1-luc的构建及表达

目的:为研究10 kb以上DNA疫苗质粒的体内电穿孔递送,构建萤光素酶报告基因表达质粒p EE14.1-luc,并验证其表达。方法:从质粒p GL3-CMV中通过酶切、补平和纯化的方法获得萤光素酶基因luc,克隆入p EE14.1载体,构建重组表达质粒p EE14.1-luc,瞬时转染293T细胞,采用Western印迹、流式细胞术和免疫荧光等方法对萤光素酶基因在体外的表达情况进行验证,并运用活体成像仪检测萤光素酶基因在小鼠活体内的表达。结果:经菌液PCR鉴定和测序验证,p EE14.1-luc与预期设计完全一致;流式细胞术检测luc阳性表达率为22.41%,免疫荧光检测可见绿色荧光表达,Western印迹检测在相对分子质量为62×103处显现目的蛋白条带;同时,利用活体成像技术也检测到萤光素酶基因在小鼠活体内的表达。结论:p EE14.1-luc表达质粒构建成功,为研究DNA疫苗体内表达机制和体内电穿孔递送条件优化奠定了基础。