Cell poration and cell fusion using an oscillating electric field.

It has been shown in previous studies that cell poration (i.e., reversible permeabilization of cell membrane) and cell fusion can be induced by applying a pulse (or pulses) of high-intensity DC (direct current) electric field. Recently we suggested that such electro-poration or electro-fusion can also be accomplished by using an oscillating electric field. The DC field relies solely on the dielectric breakdown of the cell membrane to induce cell fusion. The oscillating field, on the other hand, can produce not only a dielectric breakdown, but also a sonicating motion in the membrane that could result in a structural fatigue. Thus, a combination of a DC field and an oscillating field is expected to enhance the efficiency of cell poration and cell fusion. This study is an experimental test of such an idea. Here, pulses of high-intensity, DC-shifted RF (radio frequency) electric field were used to induce cell poration and cell fusion. The fusion experiments were done on human red blood cells. The poration experiments were done on a fibroblast cell line using a molecular probe (which is a DNA plasmid containing the marker gene chloramphenicol acetyltransferase, CAT) and assayed by a gene transfection technique. It was found that the pulsed RF field is highly efficient in both cell fusion and cell poration. Also, in comparison with electro-poration using a DC field, the RF field results in a higher percentage of cells surviving the exposure to the electric field.     

Facilitated electrofusion of vacuolated x evacuolated oat mesophyll protoplasts in hypo-osmolar media after alignment with an alternating field of modulated strength

Electrofusion of evacuolated and vacuolated oat leaf protoplasts is difficult because of the different size and density of these cells which results in separation of the two fusion partners during dielectrophoresis. The fusion yield of this cell system was considerably enhanced by electrofusion in hypo-osmolar media containing 0.4 M mannitol, 0.1 mM calcium acetate and 0.1% bovine serum albumin. This increase in yield was only achieved if the dielectrophoretically induced membrane contact between the two fusion partners was enhanced by an initial short 'burst' of higher field strength (500 V/cm, peak to peak, for 5 s followed by a reduction of to 90 V/cm, peak to peak, for 20 s, frequency 1 MHz). Due to the high field strength of the alternating field at the beginning of cell chain formation separation of fusion partners of different size and density was mainly avoided. Simultaneously, the short duration of this high field 'burst' avoided the generation of lethal effects in the cell membranes. The subsequent low field strength of the alternating field was sufficient to keep the aligned cells in position. Optimum fusion was induced by a single square pulse of 750 V/cm and 30 musec duration. The time required for rounding up of the heterologous fusion products decreased with decreasing osmolarity. Fusion resulted in a 5.7 +/- 1.2% yield of heterologous fusion products (compared to 0.7% using the conventional electrofusion protocol) as determined by flow cytometric assay. About 50% of the vacuolated oat protoplasts and 20-50% of the heterologous fusion products regenerated their cell walls within 5 days after hypo-osmolar treatment, but no cell divisions could be observed. Evacuolated oat protoplasts died after 2-3 days in culture without any detectable cell wall regeneration.     

细胞电融合技术及最新进展

细胞电融合（cell electrofusion）是一种发展迅速的细胞工程技术,在细胞融合研究领域得到了最广泛的应用。细胞电融合利用细胞在相对电极之间的介电电泳,诱导细胞按特定方向排列,通过电极间产生的较高场强的电脉冲使相互接触的细胞发生电穿孔,进而发生电融合。融合后的细胞得到了不同细胞的遗传物质,具有新的遗传或生物特性。目前,细胞电融合技术对生物医学、农业等相关领域的研究具有非常重要的意义。本文介绍了细胞电融合技术及其最新研究动态,并简单介绍了本实验室在该领域的研究进展。     

Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.     

葡萄糖浓度对细胞融合效果的影响

目的:细胞融合是细胞生物学领域近30年来得到迅速发展的一项新兴技术手段,因其操作简便、人工可控等优点在研究核质互作、肿瘤发生、疫苗研发和培育新型生物品种等方面均有广泛应用。其中,利用聚乙二醇(PEG)进行化学融合是细胞融合中最为常用且简便的技术手段。PEG化学融合效果受到多种因素影响,如PEG浓度、Ca2+、Mg2+、pH值等,然而对于糖类物质在细胞融合中的影响未见报道。本文旨在为了更全面了解PEG法诱导的化学细胞融合,通过优化融合条件以提高化学细胞融合效率。方法:选取鸡血血细胞为材料,通过改变原Hanks缓冲液中葡萄糖浓度,观察比较各组细胞融合率,探究葡萄糖浓度在化学细胞融合中的影响,并通过对比结果获得了对于鸡血血细胞应采用的最适葡萄糖浓度区间。结果:对于鸡血血细胞融合实验,葡萄糖浓度在10-14 mmol/L范围内细胞融合效率较原Hanks液配方高2倍左右。结论:葡萄糖对细胞融合效果具有一定的影响,可以通过调节葡萄糖浓度提高细胞融合率,从而为PEG化学细胞融合提供一种更为优化的方案。     

Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20–50 μs). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occured in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.     

Pseudo Color Fusion of Infrared and Visible Images Based on the Rattlesnake Vision Imaging System

Image fusion is a key technology in the field of digital image processing.In the present study,an effect-based pseudo color fusion model of infrared and visible...     

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

Differentiated cells can be experimentally reprogrammed back to pluripotency by nuclear transfer, cell fusion or induced pluripotent stem cell technology. Nuclear transfer and cell fusion can lead to efficient reprogramming of gene expression. The egg and oocyte reprogramming process includes the exchange of somatic proteins for oocyte proteins, the post-translational modification of histones and the demethylation of DNA. These events occur in an ordered manner and on a defined timescale, indicating that reprogramming by nuclear transfer and by cell fusion rely on deterministic processes.     

Endosperm development after fusion of isolated, single maize sperm and central cells in vitro

We demonstrate here the possibility of endosperm development in vitro after the fusion of pairs of an isolated sperm and an isolated central cell of maize. The occurrence of karyogamy and the time course of the fusion of sperm and central cell nuclei are presented. The fusion of the sperm nucleus occurred either with one of the two polar nuclei or with the secondary nucleus and was completed within 2 hr after in vitro cell fusion. The in vitro study of early events after cell and nuclear fusion indicates that the resulting primary endosperm cell develops into a characteristic tissue capable of self-organization apart from the mother tissue. The technology presented here opens the way for new cellular and molecular studies, especially of early events after sperm and central cell fusion. These studies should lead to a better understanding of the processes of double fertilization and endosperm development.     

融合酶的设计和应用研究进展

酶的分子改造和重新设计是解决酶催化工业应用瓶颈的重要途径。基于融合蛋白设计的融合酶技术是分子酶工程的一个研究热点,已逐渐应用于多功能酶和酶靠近效应的构建与控制研究中,显示出重要的理论和应用研究价值。文中对近年来融合酶的分子设计策略和应用研究的进展进行了综述。首先介绍了融合酶的概念和特点,并对最近研究中出现的融合酶构建策略进行了归纳总结,重点阐述了不同种类连接肽对融合酶的影响及其可能机理。同时,对目前融合酶的应用研究进行了归纳和讨论。最后,结合本实验室的研究,指出了融合酶领域的关键问题并对其发展方向进行了探讨和展望。     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨,使用木质纤维素生产燃料乙醇已具有重要的实践意义.木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖,传统乙醇生产菌株酿酒酵母不能利用木糖,这为使用以木质纤维素为原料发酵生产乙醇带来了困难.多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇.本文主要介绍这方面的研究进展.     

空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。     

Mg^2＋浓度对细胞融合效果的影响

目的：细胞融合是细胞生物学中一种常用的技术,有着广泛的应用,如单克隆抗体制备,核质研究,疫苗研发等。其中,聚乙二醇（PEG）化学融合是最为常用的一种细胞融合技术,影响PEG化学细胞融合效果的因素有很多,但是对一些具体因素的研究的不是很全面。本文旨在为了更全面的了解PEG诱导的化学细胞融合的影响因素,优化融合条件,以此扩大PEG化学融合应用范围。方法：以鸡血血红细胞为材料,通过调节已有的Hanks融合液中镁离子浓度,比较各实验组以及对照组的细胞融合率,探究了Mg^2＋浓度对细胞融合效果的影响,确定了为提高细胞融合效率应使用的Mg^2＋浓度区间。结果：可以在原有的Hanks配方的基础上,调节Mg^2＋浓度至10 mmol/L-20 mmol/L这个范围,细胞融合率较大。结论：Mg^2＋对细胞融合有一定影响,通过调节Mg^2＋浓度至上述合适区间可以达到较高的细胞融合率,从而为PEG化学融合提供了一种优化方案。     

代谢木糖产乙醇的酿酒酵母工程菌研究进展

随着能源价格的持续上涨, 使用木质纤维素生产燃料乙醇已具有重要的实践意义。木糖是多数木质纤维素水解产物中含量仅次于葡萄糖的一种单糖, 传统乙醇生产菌株酿酒酵母不能利用木糖, 这为使用以木质纤维素为原料发酵生产乙醇带来了困难。多年以来人们试图通过基因工程和细胞融合等方法对其进行改造使其能够代谢木糖生产乙醇。本文主要介绍这方面的研究进展。     

Efficiency of cloned embryo production using different types of cell donor and electric fusion strengths in goats

The type of somatic cell used as a cell donor and the electric field strength (EFS) applied for membrane fusion of the reconstructed oocytes are the two important aspects that need to be standardized for somatic cell nuclear transfer (SCNT). In the present study two somatic cells types, namely fibroblast cell grown from ear tissue biopsies of Barbari female goats and cumulus cells were used as somatic donor cells. For fusion of oocyte reconstructed membranes following somatic cell transfer, a dc current of 3 electrical field strength (EFS), i.e., 1.0–1.5; 2.0–2.5; 3 and above 3, were applied. When cumulus cells were used as a nuclear donor, a maximum fusion rate of (55.4 ± 3.9%) was obtained by applying 2.0–2.5 kV/cm dc current. The fusion rate obtained was significantly (P < 0.05) higher than all the other EFSs treatments of cumulus, as well as fibroblast cell types. The maximum fusion rate (31.9 ± 2.4%) for the fibroblast cell line was observed when an EFS of 2.0–2.5 kV/cm was applied. It could be concluded that the difference in membrane surface properties between the cumulus and fibroblast cell may contribute to the higher fusion rate obtained in cumulus cells for cloned embryo production.     

Cell hybridization by electrofusion on filters

Electric field pulses induce permeabilization and associated fusogenicity in cell membranes. Electrofusion of cells is usually performed in two steps: the first is the creation of close intercellular contacts; the second is an application of electric pulses that induces membrane fusion. Very large cell contacts can be obtained by a filter aspiration method. A cell monolayer is created by controlled suction on biocompatible filter. No spontaneous fusion results. Just after filtration, electrofusion is obtained by field pulses applied parallel to the filter. Cell viability is not strongly affected and cells recover their spherical shape in the minute time range after filtration. The electrical parameters, the cell density, and the flow rate control fusion. Fusion is obtained with cells of different origins with very different adhesion properties. Hybrid cells are easily formed. This approach appears to be a very efficient method for cell hybridization with an easy-to-use protocol.     

Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

Membrane skeleton restraint of surface shape change during fusion of erythrocyte membranes: evidence from use of osmotic and dielectrophoretic microforces as probes.

The role of the spectrin-based membrane skeleton in cell fusion was studied by following the condition-dependent diameter versus time expansion signature of the fusion zone in electrofused pairs of erythrocyte ghost membranes. Previous work showed that the presence of the dielectrophoresis-inducing alternating electric field, which is used to bring membranes into contact through pearl chain formation, had a detectable promoting effect on fusion zone expansion. Two new dielectrophoresis protocols were used in the present work to utilize this externally generated and controllable microforce field to probe the forces intrinsic to the system that drives the expansion of the fusion zone. First, fusion zones expanded to a greater diameter in a strong AC field compared to a weak AC field, and they expanded to a greater diameter if erythrocyte ghosts received a prior heat treatment (42 degrees C, 20 min). Furthermore, flat diaphragm fusion zones broke down into open lumen fusion zones sooner (i.e., had shorter lifetimes) when they were expanding more quickly. Second, changing the AC field strength at specific times during the fusion zone expansion led to an immediate visco-elastic response. However, shifting the AC field strength to zero after 5 s of fusion zone expansion resulted in a subsequent decrease in the average fusion zone diameter. This suggests not only that the spectrin-based membrane skeleton actually tends to prevent the rounding up process but that it may be capable of generating an antirounding force, which has broad implications for the role of the membrane skeleton in cell fusion. These results are consistent with the hypothesis that flat diaphragm fusion zones induced in heat-treated membranes were very easily stretched and that membrane-based forces that control or drive the expansion process must originate from membrane area that is outside rather than inside the fusion zone. Lastly, when an outward-directed osmotic pressure-based microforce was present at the time that erythrocyte ghosts were fused, the fusion zone diameter underwent a greater expansion in the 0-1 s interval after fusion. This suggests that an osmotic pressure-based microforce can be used to experimentally calibrate the dielectrophoretic force.     

用于抗体/抗体片段表达的系统及其高表达策略

随着诊断及治疗领域内单克隆抗体及片段的广泛应用,传统的杂交瘤生产技术已很难满足对其日益增长的需求。由于基因重组技术及生物工程技术的迅速发展及日益成熟,大规模生产单克隆抗体/片段已成为可能。对目前常用的载体及表达系统进行列举和比对,这些系统包括大肠杆菌(Escherichia coli, E.coli)系统、酵母表达系统、昆虫细胞表达系统及哺乳动物细胞表达系统。在第二部分,对提高表达产率的策略进行了探讨,其中包括:表达蛋白质本身的修饰(如蛋白融合、定点突变)、表达系统的选择、密码子优化、表达环境的优化和抗体体内表达等。最后,对抗体/抗体片段高表达的前景作了展望,认为生物信息学将在此领域发挥重大作用,并且相信建立高表达平台的时机已经来到。     

The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41?±?9?% yield, while in isotonic buffer 32?±?11?% yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1?% in isotonic buffer to 10?±?4?% in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.