A multi-channel electroporation microchip for gene transfection in mammalian cells

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.     

Efficient expression of foreign genes in CHO DHFR cellsby electroporation

DHFR-deficient Chinese hamster ovary (CHO DHFR⁻) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR⁻ cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-β-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400 V, 375 μF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR⁻ cells by electroporation.     

Survival of dissociated adrenal chromaffin cells of rat and monkey transplanted into rat brain

Summary Adrenal chromaffin cells from adult rats and monkeys were mechanically dissociated and implanted into the striatum of adult rats by stereotaxic injection. Rat chromaffin cells survived (5%) and showed differentiation by forming processes 1 h-28 days after implantation. Monkey chromaffin cells survived for 48 h but showed very little formation of processes. The method presented allows rapid nonenzymatic dissociation and transplantation of adrenal medullary cells.     

Efficient removal of LoxP-flanked genes by electroporation of Cre-recombinase mRNA

Introduction of Cre-recombinase in target cells is currently achieved by transfection of plasmid DNA or by viral-mediated transduction. However, efficiency of non-viral DNA transfection is often low in many cell types, and the use of viral vectors for transduction implies a more complex and laborious manipulation associated with safety issues. We have developed a non-viral non-DNA technique for rapid and highly efficient excision of LoxP-flanked DNA sequences based on electroporation of in vitro transcribed mRNA encoding Cre-recombinase. A K562-DSRed[EGFP] cell line was developed in order to measure Cre-mediated recombination by flow cytometric analysis. These cells have a stable integrated DSRed reporter gene flanked by two LoxP sites, and an EGFP reporter gene, which could only be transcribed when the coding sequence for DSRed was removed. The presented data show recombination efficiencies, as measured by appearance of EGFP-fluorescence, of up to 85% in Cre-recombinase mRNA-electroporated K562-DSRed[EGFP] cells. In conclusion, mRNA electroporation of Cre-recombinase is a powerful, safe, and clinically applicable alternative to current technologies used for excision of stably integrated LoxP-flanked DNA sequences.     

GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens

The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as “presynaptic autoreceptors” in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α₂ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well.     

Transfection of Schistosoma mansoni by electroporation and the description of a new promoter sequence for transgene expression

We sought to investigate the efficacy of electroporation for the introduction of plasmid-based DNA constructs into Schistosoma mansoni, and expanded our study to examine parameters governing transgene expression, including requirements of a 5′ and 3′ flanking sequence, as well as parasite developmental effects on transgene expression. We used luciferase as a reporter gene for this application. Our data show that electroporation allows the transfection of immature schistosomes, and defines 5′ promoter sequence from the schistosome actin gene (SmAct1.1), coupled promiscuously with various 3′ terminator sequences, as a powerful promoter of transgene expression in growing, but not early non-growing, schistosomula. The methodology described herein will facilitate ectopic expression of genes of interest in schistosomes.     

Efficient transformation of Legionella pneumophila by high-voltage electroporation

A simple and reproducible method has been developed to transform Legionella pneumophila by electroporation. Effects of different conditions, including electric field strength, pulse length, DNA quality and cell density, were evaluated. Using our method, an efficiency of up to 6 x 10(7) transformants/microg DNA was obtained. This optimized transformation procedure should efficiently facilitate gene manipulations in L. pneumophila, such as plasmid transfer, transposon mutagenesis, library transformation for complementation cloning, etc.     

In vivo plasmid DNA electroporation resulted in transfection of satellite cells and lasting transgene expression in regenerated muscle fibers

In vivo plasmid DNA electroporation resulted in elevated and lasting transgene expression in skeletal muscles. But the nature of the cells that contributed to sustained gene expression remains unknown. We followed the fate of plasmid DNA delivered with electroporation and systematically investigated the time course and location of transgene expression in muscle tissues both with GFP and luciferase. Furthermore, satellite cell activation after electroporation was confirmed by RT-PCR and immunohistochemistry analysis. The activated satellite cells were shown to be able to uptake the injected plasmid DNA and express transgene products as regenerated myocytes. We found that cells with longer gene expression durations were mostly regenerated muscle fibers. In contrast, expression in pre-existing muscle fibers was rather transient. We also presented in this study that immune response to transgene products might hamper the lasting gene expression. Based on these observations, we proposed that the underlying mechanism for prolonged transgene expression in the muscles after electroporation is related to the activation and transfection of myogenic satellite cells which subsequently developed into regenerated muscle fibers.     

Efficient transfection of primary zebrafish fibroblasts by nucleofection

Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.     

Transformation of Bacillus thuringiensis vegetative cells by electroporation

A highly efficient procedure for the transformation of Bacillus thuringiensis and Bacillus subtilis using covalently closed circular plasmid DNA was developed by using the small Staphylococcus aureus plasmid pC194 and electroporation. We have achieved transformation efficiencies in B. thuringiensis subsp. kurstaki (HD-73) greater than 5 x 10(6) transformants/micrograms plasmid DNA. The electro-transformation (or electroporation) procedure also worked with B. subtilis 168 although at a 200-fold less level of efficiency. The results indicated that the plasmid exists in double and single-stranded forms both in B. subtilis and B. thuringiensis. A second single-stranded species was also observed in both species. This technique may prove to be applicable to other members of the genus Bacillus.     

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of ±1.5% of full scale in 65 536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 μm when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 · 10^?4 torr^?1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 · 10^?4 torr^?1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficent of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.     

High efficiency electroporation of intact Corynebacterium glutamicum cells

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.     

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics and determinants of osmotic lysis in chromaffin granules

(1) Using isolated bovine chromaffin granules, we demonstrate that osmotic lysis is not a random process and establish the osmotic pressure dependence of osmotic lysis in chromaffin granules, the so-called osmotic fragility curve. (2) We show by measuring the release of constituents of the granule core and correlating these with changes in spectroscopic parameters (turbidity and endogenous catecholamine fluorescence), that the latter can be safely used to measure lysis. (3) Within a particular granule population, noradrenaline granules lyse at higher osmolarities than adrenaline granules, suggesting a higher core osmolarity of the noradrenaline granules. (4) The size distribution of chromaffin granules as a function of lysis was determined by the use of whole mount electron microscopy. It is shown that the mean size of chromaffin granules decreases as a function of lysis. (5) On the basis of theoretical considerations three alternative models of the sequence of osmotic lysis in chromaffin granules are proposed. The experimental results best support a model which postulates that during partial osmotic lysis, granule membranes reseal into smaller vesicles after graded release of contents. The osmotic fragility would represent several cycles of lysis and resealing and would not be a reflection of the distribution of osmotic pressures in the granule population.     

Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix

Myogenic cell lines have been used widely in the study of myogenic differentiation, muscle regeneration and homeostasis, but, myoblasts and myotubes are difficult to transfect using conventional techniques. We have used liposome-based transfection method to introduce a green fluorescence protein (GFP)-expressing plasmid into Matrigel basement membrane matrix-coated C2C12 mouse myoblast cells. Myoblasts adhered and proliferated more rapidly on a Matrigel; thus, a dramatic increase in transfection efficiency can be obtained compared to Matrigel-untreated cells. Transfection efficiency was determined by counting fluorescent and total cells from six random fields for each condition. This protocol results in efficient (up to 60–70%) transfection of C2C12 myoblasts, high levels of GFP expression and low rate of cell death (10%). This technique is rapid, reliable, uses a lipid-based transfection reagent, and yields high transfection rates in a previously hard-to-transfect cell type.     

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Under low stimulation, adrenal chromaffin cells release freely soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.     

The processed pseudogene of mouse thymidine kinase is active after transfection

Aside of the gene coding for cytoplasmic thymidine kinase, the genome of mouse cells carries two pseudogenes. Both are inactive in situ. One of the pseudogenes is a processed pseudogene in which a two base pair deletion caused a shift of the reading frame and a shortening of the gene product from the 233 amino acids of thymidine kinase to 177 amino acids in the pseudogene product. We report here that introduction of this pseudogene into LTK⁻ cells gave rise to cells with a thymidine kinase positive phenotype. The transformed cells carried multiple copies of the pseudogene the upstream region of which exhibited low but measurable promoter activity. Replacement of the upstream region of the pseudogene by a promoter of Simian virus 40 or of the mammary tumor virus resulted in high transfection efficiencies and in cell lines exhibiting high thymidine kinase activities.     

A novel transfection method for eukaryotic cells using polyethylenimine coated albumin microbubbles

Albumin microbubbles have been intensively studied for their application in gene delivery. However, with negative surface potential, albumin microbubbles hardly bind plasmid DNA, which might contribute to their low transgene efficiency. In this study, we developed polyethylenimine (PEI) coated albumin microbubbles (PAMB) which were prepared by sonicating the mixture of human albumin, PEI, polyethylene glycol and glucose. CHO cells, COS cells and 293T cells were transfected with PEI, PEI + albumin, PAMB and Lipofectamine 2000, respectively. Our results showed that the surface potential was elevated and PAMB could bind plasmid DNA. The transgene efficiency of PAMB was higher than PEI and PEI + albumin (P < 0.05), and PAMB performed the same transgene effect as Lipofectamine 2000 did but with lower cytotoxicity than Lipofectamine 2000. Albumin microbubbles modified by PEI has high transgene efficiency and low cytotoxicity even without ultrasound medication, making it a useful non-virus gene delivery method in vitro.     

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb Cla I/ Sph I chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum , SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.