Nucleofection is a valuable transfection method for transient and stable transgene expression in adipose tissue-derived stem cells

Adipose tissue-derived stem cells are a powerful tool for in vitro study of adult stem cell biology. So far, they have not been extensively used for gain or loss of function studies since they are resistant to most common transfection methods. Herein, we tested several classic transfection methods on human multipotent adipose tissue-derived stem (hMADS) cells. Our results showed that lipofectants and calcium phosphate were poorly efficient for transgene delivery in hMADS cells. In contrast, nucleofection, an electroporation-based method that is assumed to target plasmid DNA directly to the cell nucleus, led to a significant transient transgene expression in hMADS cells (up to 76% enhanced green fluorescent protein [EGFP]-positive cells were detected). Furthermore, after selection of hMADS cells that were nucleofected with a selectable plasmid coding for EGFP, stable EGFP expressing clones could be propagated in culture and efficiently induced to differentiate into EGFP-positive adipocytes and osteoblasts. Finally, we verified that nucleofected hMADS cells could produce a functional, transgene-encoded, secreted protein. To this aim, hMADS cells were nucleofected with a plasmid coding for leukemia inhibitory factor (LIF). This protein was detected at high concentrations in supernatants from pCAG-LIF transfected hMADS cells. Moreover, supernatants were able to maintain mouse embryonic stem cells' undifferentiated phenotype, indicating that hMADS cells could secrete a functional LIF protein. Taken together, our data demonstrate that nucleofection allows both transient and stable gene expression in adipose tissue-derived stem cells, without impairing their differentiation potential.     

核转染对成体骨髓源性干细胞的影响

背景：成体骨髓源性干细胞是实施细胞治疗的重要细胞来源。对成体骨髓源性干细胞的示踪,是研究其移行、分化的规律与机制并进一步阐明再生潜能、疗效的关键。 目的：探讨经转染后的成体骨髓呈克隆样干细胞,在细胞表型、增殖能力、心肌分化潜能是否存在的影响。 方法：应用核转染技术利用U-23程序将编码maxGFP报告基因的载体对成体骨髓呈克隆样干细胞进行转染,应用MTT法对核转染前后的生长曲线进行测定,使用3 μmol/L 5-氮胞苷对核转染前后成体骨髓呈克隆样干细胞进行向心肌分化诱导,并利用RT-PCR对向心肌分化的特异性标记物GATA4与MLC-2v的表达进行测定。使用成年SD大鼠心肌梗死左前降支结扎模型,对使用经maxGFP转染的成体骨髓呈克隆样干细胞施心内注射并于注射后的第2天和第7天对经注射心脏行冰冻切片并观察maxGFP在体内的表达情况。 结果与结论：经核转染24 h后,第47代及第119代成体骨髓呈克隆样干细胞的转染率为49.4%和43.1%,转染后5 h,开始出现呈绿色荧光阳性的细胞,经核转染后仍能保持小圆球形、可贴壁、呈克隆样生长。MTT检测结果显示：核转染前后第47代及第119代均具有相似的生长曲线。核转染前第47代及119代细胞平均群体倍增时间分别为8.57,10.28 h;核转染后分别为9.42,10.42 h,各代前后比较差异无显著性意义(P=0.551,P=0.774)。RT-PCR检测结果显示：核转染前5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强;核转染后5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强,上述2个向心肌分化指标于转染前后变化并不明显。体内实验结果：心内注射经核转染后的成体骨髓呈克隆样干细胞,第2天及第7天可在经注射心肌中发现有少量绿色荧光阳性的细胞。提示,核转染可快速地将外源基因导入细胞,经核转染后的成体骨髓呈克隆样干细胞仍能保持其细胞形态、增殖能力及向心肌分化潜能,然而,经maxGFP基因核转染的成体骨髓呈克隆样干细胞,移植入心肌后只有少数细胞能表达经转染基因。     

Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Fast and Efficient Transfection of Mouse Embryonic Stem Cells Using Non-Viral Reagents

Reliable and efficient DNA and RNA transfection methods are required when studying the role of individual genes in mouse pluripotent stem cells. However, these cells usually grow in tight clusters and are therefore more difficult to transfect than many other cell lines. We have found that transfection is especially challenging when mouse embryonic stem (mES) cells are cultured in the newly described 2i medium, which is based on two chemical inhibitors of differentiation pathways. In the present study we have performed a side-by-side comparison of commercially available, non-viral transfection reagents with regard to their ability to deliver plasmid DNA and siRNA into adherent and/or trypsinized mES cells cultured in 2i medium, assessing transfection rates, plasmid gene expression, siRNA mediated knockdown of Oct4 and viability. Finally, we present a fast and efficient method for transfection of trypsinized mES cells using the liposomal-based Lipofectamine 2000. With only a five-minute long transfection time we obtained at least 85 % transfected cells with 80 % maintained viability. Moreover, this protocol saves up to a day of experimental time since the cells are in suspension at the time of transfection, which allows for immediately re-plating into the appropriate format. This fast, simplified and highly efficient transfection method will be valuable for both basic research and high-throughput applications.     

Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells

Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow- derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7%+/-2.9% and 42.5%+/-3.4%, respectively. Cell recoveries and viabilities were 38.7%+/-2.9%, 44.5%+/-3.9% and 91.4%+/-1.3%, 94.31%+/-0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4%+/-2.9% (U-23) and 16.6%+/-1.4% (C-17) compared with 3.6%+/-2.4% and 5.4%+/-3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p<.005 for all comparisons). Nucleofection did not affect the immunophenotype of hM-SCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents.     

人胰岛素样生长因子1基因转染脂肪源性干细胞的效应

背景：人胰岛素样生长因子1基因对脂肪源性干细胞的增殖和分化也可能会产生有效作用。 目的：验证人胰岛素样生长因子1基因转染对体外培养的脂肪源性干细胞的效应。 方法：构建含人胰岛素样生长因子1基因的双顺反子真核表达载体pIRES2-EGFP-hIGF-1,利用阳离子脂质体Lipofectamine 2000介导转染体外培养的人脂肪源性干细胞。观察基因转染后细胞增殖及形态的变化,倒置荧光显微镜观测标记基因增强绿色荧光蛋白的表达并计算转染效率,酶联免疫吸附试验检测培养上清中人胰岛素样生长因子1的浓度,免疫组织化学染色及RT-PCR检测人胰岛素样生长因子1的表达,流式细胞仪检测转染前后细胞周期的变化。 结果与结论：测序及酶切证实真核表达载体pIRES2-EGFP-hIGF-1构建正确。体外培养的脂肪源性干细胞为多种形态并存,转染后6 h检测到有EGFP的表达,至60 h达到高峰,转染效率为(16±3)%。细胞上清中人胰岛素样生长因子1的浓度在60 h达到22.65 μg/L。免疫组织化学染色及RT-PCR均检测到人胰岛素样生长因子1的表达。转染后的细胞分裂增殖加快,细胞群体倍增时间缩短,S期细胞比例增多。证实人胰岛素样生长因子1基因可有效转染脂肪源性干细胞并表达人胰岛素样生长因子1蛋白质,同时可促进细胞增殖。     

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers

Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.     

重组pcDNA3.1-hBMP-2转染兔脂肪干细胞体外诱导的成骨分化

背景：骨形态发生蛋白2具有很强的诱导干细胞成骨活性。 目的：构建人骨形态发生蛋白2基因真核表达载体,探讨其转染脂肪干细胞后的成骨效果。 方法：通过噬菌斑原位杂交筛选人混合细胞cDNA文库获得人骨形态发生蛋白2基因,与真核表达载体pcDNA3.1-连接,构建重组质粒pcDNA3.1-hBMP-2。利用脂质体Lipofectamine™ 2000分别介导人骨形态发生蛋白2 基因、EGFP基因转染第4代脂肪干细胞,并经G418进行筛选。 结果与结论：酶切鉴定及DNA测序结果证实重组质粒pcDNA3.1-hBMP-2构建成功。经计算脂质体介导的脂肪干细胞瞬时转染率为(18.0±0.42)%,并经过G418筛选后获得了稳定转染的细胞。细胞生长曲线表明转染后对脂肪干细胞生长、增殖无明显影响。ELISA检测发现人骨形态发生蛋白2组的人骨形态发生蛋白2因子表达量均高于EGFP组及未转染组,且能够稳定表达。经人骨形态发生蛋白2基因转染的脂肪干细胞的Ⅰ型胶原含量、碱性磷酸酶活性以及钙结节数目均比EGFP组及未转染组有明显升高。     

小鼠pdx-1基因真核表达载体的构建及其在胚胎干细胞中的表达

目的： 克隆小鼠pdx-1基因，构建其真核表达载体，并在小鼠胚胎干细胞中表达，为糖尿病的细胞移植治疗奠定基础。方法： PCR扩增小鼠胰腺pdx-1基因 cDNA，酶切后和携带绿色荧光蛋白报告基因的真核表达载体pEGFP-N1重组，将pdx-1基因 cDNA片段连接到pEGFP-N1载体的多克隆位点，形成重组载体pEGFP/pdx-1，转化大肠杆菌DH5α菌株，构建成pdx-1基因真核表达载体质粒。扩增DH5α后抽提质粒DNA，Hind Ⅲ 和BamHⅠ酶切，电泳，DNA测序鉴定。鉴定正确的质粒DNA用脂质体包裹后转染小鼠胚胎干细胞MESPU13。结果： 从小鼠胰腺cDNA扩增出876 bp的DNA片段并成功重组到pEGFP-N1载体中。经酶切和DNA测序验证，插入载体的DNA片段为pdx-1基因，插入方向正确。重组质粒经脂质体转染胚胎干细胞MESPU13，24 h 后观察到绿色荧光蛋白报告基因和目的基因的pdx-1表达。结论： 小鼠pdx-1基因的克隆和真核表达载体构建获得成功，为进一步研究其功能奠定了基础。     

增强型绿色荧光蛋白基因转染小鼠胚胎干细胞及其无血清神经细胞诱导

目的构建表达绿色荧光蛋白的小鼠胚胎干细胞(MES)克隆，观察基因转染对MES细胞增殖和无血清诱导为nestin阳性神经前体细胞(NPCs)的影响。方法原代分离小鼠胚胎成纤维细胞并经丝裂霉素-C处理作为饲养层细胞。质粒的转化、抽提、纯化。电穿孔法基因转染，MES细胞克隆的NBT／BCIP染色，NPCs的nestin免疫组织化学染色。结果EGFP基因转染后可得到表达绿色荧光蛋白的MES细胞克隆，采用N2或ITSF无血清条件培养基可将其定向诱导为nestin阳性的并能发绿色荧光的NPCs。基因转染后的MES细胞增殖、神经前体细胞诱导率和未转染组相比均无明显下降，但NPCs绿色荧光的表达较诱导前明显下降。结论基因转染对ES细胞的增殖和神经分化无明显影响。     

A gene transfection for rat mesenchymal stromal cells in biodegradable gelatin scaffolds containing cationized polysaccharides

The objective of this study was to design three-dimensional scaffolds of bone marrow-derived stem cells capable of their gene transfection and evaluate the transfection extent. Three-dimensional scaffolds of gelatin and β-tricalcium phosphate (β-TCP) were prepared. Spermine-introduced pullulan (spermine-pullulan) was prepared as the non-viral carrier of gene transfection. The spermine-pullulan was mixed with a luciferase plasmid DNA to prepare their polyion complex. The scaffolds were treated with succinylated gelatin (suc-gel) at different concentrations, treated in different methods of freeze-drying and dehydrothermal treatment, and placed in the aqueous of complexes to prepare various scaffolds containing the complex. When the stem cells were seeded into the scaffolds to evaluate the gene transfection of cells, the level of plasmid DNA transfection depended on the method of complex containing scaffolds preparation. The complexes were released with time from the scaffolds although the release profile depended on the type of scaffolds. The order of gene transfection for the stem cells in the scaffolds was in good accordance with that of plasmid DNA released. It is possible that cells were transfected with the complexes released from the scaffolds.     

High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer

Dendritic cells (DCs) are bone marrow-originated, professional antigen-capturing cells and APCs, which can function as vaccine carriers. Although efficient transfection of human DCs has been achieved with viral vectors, viral gene products may influence cellular functions. In contrast, nonviral methods have generally resulted in inefficient gene transfer, low levels of gene expression, and/or low cell viability. Monocyte-derived DCs are the most common source of DCs for in vitro studies and for in vivo applications. We hypothesized that reduction of the time to generate immature DCs (iDCs) might result in higher viability after transfection. Therefore, we established a protocol to generate human iDCs from CD14(+) monocytes within 3 days. These "fast" iDCs were phenotypically and functionally indistinguishable from conventional iDCs, showing high endocytic ability and low antigen-presenting capacity. Furthermore, the fast iDCs matured normally and had similar antigen-presenting capacity to conventional mature DCs. To optimize transfection of iDCs, we compared nonviral transfection of plasmid DNA and in vitro-transcribed (IVT) RNA with transfection reagents, electroporation, and nucleofection. Nucleofection of IVT RNA with the X1 program of an Amaxa Co. Nucleofector resulted in the most efficient transfection, with an average of 93% transfected iDCs, excellent long-term viability, and strong protein expression. Furthermore, the IVT RNA-transfected iDCs retained all phenotypic and functional characteristics of iDCs. This method is applicable to most purposes, including in vitro functional assays, in vivo DC immunotherapy, and DC-based vaccines.     

Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells

The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically.     

两种传代方法影响人胚胎干细胞转染效率的比较

背景:现有方法将外源分子如DNA导入到人胚胎干细胞用于科学研究的效率普遍较低,如何优化现有条件,提高转染效率显得尤为重要。目的:比较两种不同的传代方法对人胚胎干细胞系H9转染效率的影响,优化胚胎干细胞转染条件。方法:人胚胎干细胞系H9分别采用小克隆传代法和单细胞传代法进行传代,传代后继续培养细胞48 h,用Lipofectamine 3000转染pAdTrack-AKT1荧光质粒2 d后,荧光显微镜下观察荧光质粒的表达,流式细胞仪检测人胚胎干细胞的转染效率;RT-qPCR和Western blot分别检测转染后AKT1在mRNA和蛋白质水平的表达。结果与结论:①荧光显微镜下观察发现单细胞传代组表达荧光质粒的细胞数量更多,流式细胞仪检测单细胞传代法的转染效率[(47.18±2.00)%]高于小克隆传代法的转染效率[(19.52±0.86)%],差异有显著性意义(P<0.01);②单细胞传代组转染后AKT1 mRNA和蛋白的表达均高于小克隆传代组,差异有显著性意义(P<0.01);③结果表明,采用单细胞传代法,增加细胞与转染试剂脂质体的接触面积可提高人胚胎干细胞的转染效率。     

重组人肝细胞生长因子在人脐带间质干细胞中高效表达

目的：构建能表达人肝细胞生长因子(hHGF) 的pWPI-hHGF载体，并将含hHGF基因的重组慢病毒载体转染人脐带间质干细胞（hUCMSCs），观察人脐带间质干细胞中hHGF的表达情况。方法：将携带目的基因hHGF的 pUC-SRα/hHGF质粒亚克隆到真核细胞表达载体pWPI载体上，构建重组质粒pWPI-hHGF。基因测序进行HGF的鉴定。用磷酸钙沉淀法，将pWPI-hHGF、pAX2、pMD2G共同转染包装细胞293T细胞，获得携带目的基因hHGF和GFP基因的重组慢病毒pWPI-hHGF。将pWPI-hHGF及阳性对照质粒pWPI-GFP分别用Lipofectamin 2000介导转染体外培养的hUCMSCs。在荧光显微镜下计数，确定阳性对照质粒的转染数，从而估计该基因的转染效率。蛋白印迹法检测HGF和GFP蛋白的表达，ELISA法检测细胞上清液hHGF含量。结果：DNA 测序显示hHGF基因成功地插入到pWPI载体中。包装细胞293T转导pWPI-HGF质粒后，转染阳性率达100％。阳性对照质粒转染人脐带间质干细胞24 h后，在荧光显微镜下观察，其转染效率达80%以上。蛋白印迹法检测靶细胞中hHGF表达呈强阳性，而对照组表达量极低，两者差异显著(P<0.01)。检测收集转染hHGF后的人间质干细胞上清液中hHGF的表达含量明显高于对照组(P<0.01)。结论：构建的在真核细胞内表达hHGF的重组质粒pWPI-hHGF转染人脐带间质干细胞，能获得hHGF基因在人脐带间质干细胞中大量、稳定的表达。     

RNA干扰Rbl-2基因调控DNA甲基化转移酶介导心脏干细胞凋亡

目的:视网膜母细胞瘤样蛋白2(Rbl-2)在调控DNA甲基化转移酶(DNMT)中发挥重要作用,而DNMT影响干细胞的分化。本文旨在探讨调控Rbl-2基因的表达对心脏干细胞凋亡的影响。方法:分离培养人心脏干细胞,转染Rbl-2 siRNA。采用real time RT-PCR检测Rbl-2和DNMT-3B mRNA表达水平,Westernblotting检测DNMT-3B蛋白表达和caspase-3活化片段,并以Annexin V-FITC/PI双染色-流式细胞术检测细胞凋亡率。结果:在人心脏干细胞中,转染siRNA后Rbl-2 mRNA表达水平与阴性对照组相比显著降低(P0.01),同时DNMT-3B mRNA和蛋白表达水平与阴性对照组相比均显著升高(P0.01),差异显著。与阴性对照组相比,转染Rbl-2 siRNA的人心脏干细胞caspase-3激活,表现为caspase-3活性片段与caspase-3前体的比值显著增高(P0.01),annexin V阳性细胞凋亡率上升(P0.05)。结论:Rbl-2基因在心脏干细胞的生存中具有正性调控作用,抑制其表达能促进心脏干细胞的凋亡,其调控机制与表观遗传学的修饰密切相关。     

转染胶质细胞源性神经营养因子诱导神经干细胞分化

背景：神经干细胞为神经系统功能重建和神经再生提供了一条新的途径,解决其定向诱导分化问题仍是目前的研究热点。 目的：探讨转染胶质细胞源性神经营养因子基因诱导大鼠胚胎神经干细胞向神经元和多巴胺能神经元分化的作用。 方法：构建PcDNA3-GDNF-GFP表达质粒,用脂质体介导该质粒转染大鼠胚胎神经干细胞并进行诱导分化,荧光显微镜观察转染情况,免疫荧光染色检测β微管蛋白Ⅲ和酪氨酸羟化酶表达。 结果与结论：胶质细胞源性神经营养因子基因转染后3 d,可观察到细胞呈绿色荧光细胞球状。诱导分化7 d,神经干细胞分化为神经元以及多巴胺神经元的比例明显增高。结果表明胶质细胞源性神经营养因子基因可以促进神经干细胞向神经元以及多巴胺能神经元分化。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system

Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.     

Efficient transfection method for primary cells

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.     

A toolbox facilitating stable transfection of Eimeria species

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.