共查询到19条相似文献,搜索用时 171 毫秒
1.
脂质体法和电穿孔法转染哺乳动物细胞研究 总被引:3,自引:0,他引:3
用脂质体法和电穿孔法分别转染Cos-7,Vero和Namalwa细胞.发现脂质体法在转染效率和操作方便方面比电穿孔法优越,而电穿孔法对细胞种类的适用性方面似乎比脂质体法广. 结果表明,电穿孔法能转染Cos-7,Namalwa和Vero细胞,而用脂质体法只能转染Cos-7和Vero细胞. 相似文献
2.
影响细胞电穿孔效率的因素 总被引:5,自引:0,他引:5
根据细胞电穿孔的原理和细胞电融合仪的性能,将细胞电融合仪用于细胞电穿孔,使Cos-7,Vero和Namalwa等三株细胞都得到了转染。转染率分别达到外43%,0.42%和1.66%。通过β-半乳糖苷酶组织化学染色确定转移基因的表达,建立了一种快速优化电转染参数的方法。该方法只须改变电压一个参数,全部实验可以在一天内完成,该方法较之其它转染方法(如磷酸钙沉淀法,脂质体法,DEAE-葡聚糖法以及逆转录病毒介导转染法)具有可重复性强,经济便宜以及适用于多种细胞系的特点。 相似文献
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《生物技术》2014,(2)
目的:采用不同的转染方法介导siRNA片段转染小鼠巨噬细胞,对不同细胞转染方法进行比较。方法:分别采用脂质体转染、罗氏转染试剂转染、电穿孔法和慢病毒介导siRNA片段转染小鼠巨噬细胞,然后通过流式细胞仪分析不同转染方法的转染效率,并测定对细胞活性的影响以及目的基因的表达水平。结果:慢病毒介导siRNA转染小鼠巨噬细胞效率最高,可达34.75±5.30%,且RNA干扰效果最好;其次为罗氏转染试剂转染和电穿孔法,但电穿孔法细胞活力最低;脂质体转染效率最低,仅为12.17±1.53%,但细胞活力最好。结论:通过对4种小鼠巨噬细胞转染方法的比较,明确了4种转染方法的优缺点,为小鼠巨噬细胞转染提供了实验依据。 相似文献
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HCV核心蛋白诱导Cos-7细胞凋亡 总被引:1,自引:0,他引:1
将丙型肝炎病毒(HCV)核心蛋白(Core)基因的全长序列插入到真核表达载体pCDNA3 CMV启动子下游,构建真核表达载体pCDNA3-Core,用脂质体LipoVecTM转染Cos-7细胞系进行瞬时表达;DNA转染24h后用免疫斑点试验检测在细胞中表达的Core蛋白;转染72h后用Hoechst染色和DNA Ladder检测Cos-7细胞的凋亡情况;荧光染色观察到了细胞凋亡核碎裂,琼脂糖凝胶电泳也呈现出180-200bp整数倍的梯形带,呈现典型的细胞凋亡特征.这些结果表明HCV Core蛋白的表达能引起Cos-7细胞凋亡,Core蛋白的这种功能可能在HCV的持续感染过程中起着一定的作用. 相似文献
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脂质体介导转染法的原理与应用 总被引:5,自引:0,他引:5
脂质体是磷脂分散在水中时形成的脂质双分子层 ,又称为人工生物膜。最初 ,人们只是运用脂质体模拟生物膜 ,研究膜的构造及功能 ,从而发现了膜的融合及内吞作用 ,因而可用作外源物质进入细胞的载体。相对于电穿孔法和磷酸钙共沉淀转染法 ,脂质体介导转染法简便易行 ,成本适中 ,具有较高的转染率和较小的细胞毒性。1 .脂质体的组成和制备1 .1 组成脂质体的脂类 现有的商业化脂质体均为阳离子脂类与中性脂类的复合体 ,如LipofectAMINE、Lipofectin等 ,中性脂类多为二油酰磷脂酰乙醇胺 (DOPE)。其中 ,阳离子脂类… 相似文献
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本研究采用腺病毒感染、慢病毒感染、脂质体转染和电穿孔转化方法将含有绿色荧光蛋白(GFP)的质粒转入经过差异贴壁法初步分离纯化的小鼠精原干细胞(SSCs)中,转染48 h后通过流式细胞仪检测GFP阳性细胞比例比较4种方法在体外转染精原干细胞的效率.结果显示,脂质体转染效率最高仅为8.64%,不能满足对精原干细胞进一步实验的要求;电穿孔法效率最高达到25.27%,但转化后细胞大量死亡;腺病毒转染细胞的效率达到了32.4%;慢病毒转染效率最高,达到74.25%. 因此,慢病毒转染法是体外转染小鼠精原干细胞的有效方法. 相似文献
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目的:建立组成型过量表达hTERT的Vero细胞系。方法:从质粒pCI-neo-hTERT酶切、分离纯化hTERT cDNA,克隆入phEF/chMAR-hyg载体中,构建重组真核表达质粒phEF/chMAR-hyg-hTERT。采用脂质体转染法转染Vero细胞,经潮霉素筛选、克隆分离培养,用RT-PCR检测转染阳性细胞克隆的hTERT mRNA表达丰度,Western blotting验证高丰度表达hTERT mRNA克隆的hTERT表达。用Cedex AS20细胞密度和活力分析系统考察过量表达hTERT Vero细胞在组织培养板中批次培养的生长特性。结果:从phEF/chMAR-hyg-hTERT转染阳性细胞中筛选出hTERT mRNA和蛋白高丰度表达的Vero细胞系(Vero-T1),Vero-T细胞在批次培养中后期的细胞密度和活力均高于野生型Vero细胞。结论:成功建立了hTERT基因组成型过量表达的Vero细胞系,为探索以组成型过量表达hTERT的技术途径改善细胞培养特性、提高病毒疫苗生产工艺水平奠定了基础。 相似文献
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目的:探讨在RNAi技术条件下,HSV-1感染Vero细胞过程中,病毒间层蛋白VP16的生物学功能。方法:用T7RNA聚合酶体外合成针对VP16 mRNA的3种T7siRNAs。用Lipofectamine 2000脂质体转染Vero细胞后接种HSV-1,感染后12、24、36、48、60和72h,分别收取实验组和对照组标本,Karber法计算病毒滴度值,绘制子代病毒一步生长曲线。结果:病毒子代一步生长曲线显示,转染siRNAs的实验组Vero细胞接种HSV-1后24h(24h pi),病毒滴度明显低于对照组;12h pi也观察到病毒滴度低于对照组的现象;siRNAs具有协同作用,三种siRNAs同时作用时干扰效果最为明显;实验组病毒滴度高峰出现时间后移12h左右。结论:HSV-1间层蛋白VP16对病毒复制、成熟起重要的生物学作用。 相似文献
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The stable transfection of immortalized Rat-1 and rat skin primary fibroblast cell lines by calcium phosphate precipitation, lipofection and electroporation methods have been examined. The lipofection method was found to be better than the other methods in terms of higher transfection efficiency and convenient use. Expression of beta-galactosidase from two different viral promoters showed that the level of transgene expression depends on the promoter strength in a particular cell type. The results presented here show that the transgene expression is extremely variable among different colonies generated from individually transfected cells. Therefore, it is necessary to examine individual colonies of cells for the production of reporter gene to obtain cell lines expressing high amounts of gene products. 相似文献
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A. Hashemi F. Roohvand M. H. Ghahremani M. R. Aghasadeghi R. Vahabpour F. Motevali A. Memarnejadian 《Cytology and Genetics》2012,46(6):347-353
Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI? and Lipofectamine? 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 ± 2.36 and 73.9 ± 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI? was 14.2 ± 0.69 and 28 ± 1.11% Huh-7 and Vero cells, respectively. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines. 相似文献
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Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). 总被引:6,自引:0,他引:6 下载免费PDF全文
Electroporation is one of the most common methods used transform mammalian cells with plasmids. This method is versatile and can be adapted to meet the requirements of many cell lines. However, sometimes the efficiency of this method is low. We demonstrate that dimethyl sulfoxide (DMSO) facilitated a better DNA uptake in four different cell lines (HL60, TR146, Cos-7 and L132). The cells were electroporated with a beta-Gal expression plasmid in a medium containing DMSO (1.25%) during, and for 24 h after the pulse. In all these cells a dramatic (up to 8-fold) increase in transfection efficiency occurred after this treatment. This method opens up the possibility of using electroporation even in cells which are difficult to transfect. 相似文献
14.
Samuel McLenachan Dan Zhang Ana Belén Alvarez Palomo Michael J. Edel Fred K. Chen 《PloS one》2013,8(12)
The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. 相似文献
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Micka B Trojaneck B Niemitz S Lefterova P Kruopis S Huhn D Wittig B Schadendorf D Schmidt-Wolf IG 《Cytokine》2000,12(6):828-833
Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction. 相似文献
16.
Toshinobu Kida Sachi Fujishima Masatoshi Matsumura Pi-Chao Wang 《Biotechnology and Bioprocess Engineering》2000,5(2):92-98
Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular
filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these
insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell
lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection
of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only
electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer
than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two
main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with
rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth
of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was
also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the
regulation of cell proliferation and differentiation. 相似文献
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Embryonic stem (ES) cells are an important tool in developmental biology, genomics, and transgenic methods, as well as in potential clinical applications such as gene therapy or tissue engineering. Electroporation is the standard transfection method for mouse ES (mES) cells because lipofection is quite inefficient. It is also unclear if mES cells treated with cationic lipids maintain pluripotency. We have developed a simple lipofection method for high efficiency transfection and stable transgene expression by employing the nonclassical nuclear localization signal M9 derived from the heterogeneous nuclear ribonucleoprotein A1. In contrast to using 20 microg DNA for 10 x 10(6) cells via electroporation which resulted in 10-20 positive cells/mm2, M9-assisted lipofection of 2 x 10(5) cells with 2 microg DNA resulted in > 150 positive cells/mm2. Electroporation produced only 0.16% EGFP positive cells with fluorescence intensity (FI) > 1000 by FACS assay, while M9-lipofection produced 36-fold more highly EGFP positive cells (5.75%) with FI > 1000. Using 2.5 x 10(6) ES cells and 6 microg linearized DNA followed by selection with G418, electroporation yielded 17 EGFP expressing colonies, while M9-assisted lipofection yielded 72 EGFP expressing colonies. The mES cells that stably expressed EGFP following M9-assisted lipofection yielded > 66% chimeric mice (8 of 12) and contributed efficiently to the germline. In an example of gene targeting, a knock-in mouse was produced from an ES clone screened from 200 G418-resistant colonies generated via M9-assisted lipofection. To our knowledge, this is the first report of generation of transgenic or knock-in mice obtained from lipofected mES cells and this method may facilitate large scale genomic studies of ES developmental biology or large scale generation of mouse models of human disease. 相似文献
18.
Nakayama A Sato M Shinohara M Matsubara S Yokomine T Akasaka E Yoshida M Takao S 《Cloning and stem cells》2007,9(4):523-534
Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer. 相似文献
19.
Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian
cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals,
or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is
well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection
methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous
and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination
levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally
induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on
homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation
yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a
greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies
using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation.
By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation.
Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination,
possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells. 相似文献