Optimization of Babesia bovis transfection methods

The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa) A comparative study among four different transfection methods: transfection of infected erythrocytes and purified merozoites with 2 or 100 microg of plasmid, using electroporation (BioRad GenePulser II) or nucleofection (Amaxa) indicates that electroporation of infected erythrocytes with 100 microg of plasmid or nucleofection with 2 microg of plasmid are the most efficient ways to transfect B. bovis parasites. The data also indicate that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 microg range), whereas the inverse is true for transfection of larger quantities (100 microg range). This information will facilitate further development of efficient stable transfection systems.     

棉铃虫细胞色素P450 CYP6B6基因启动子活性检测条件的优化

旨在用阳离子脂质体介导携带有pGL3-Basic报告基因的棉铃虫细胞色素P450 CYP6B6启动子pGL-CYP6B6-promoter重组质粒转染Sf9细胞,对启动子活性的检测条件进行优化。将长度为999 bp的棉铃虫CYP6B6基因启动子亚克隆至荧光素酶报告载体pGL3-Basic上,提取无细胞内毒素的质粒,转染处于对数生长期的昆虫细胞Sf9,通过检测荧光素酶的表达量验证启动子的活性。对转染后的检测时间、加入转染质粒的量,转染质粒与内对照质粒pRL-TK的比例分别进行了优化,进而确定检测的最优条件。结果表明,转染后的最适检测时间为24 h,转染质粒的最适用量为3.2μg,报告质粒与内对照质粒用量的比例为10 1时转染效率最高。     

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system

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.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Apoptosis induced by DNA uptake limits transfection efficiency

Electrotransfection is an effective method for transfecting lymphoid cells. However, the transfection efficiency of certain lymphoid cells is low. L1210 subclones and NFS-70 pro-B cells, which are highly refractory to various transfection methods, were used to identify the limiting factors. Cells were electrotransfected with plasmids coding for green fluorescence protein or luciferase. The luciferase expression of L1210 subclone 3-3 was found to increase 6-12 h after electroporation, but decreased significantly from 12 to 48 h. The lower level of luciferase activity at later time periods correlated with decreases in cell viability, which was shown to be due to apoptosis, as determined by propidium iodide/acrindine orange staining, DNA laddering, and prevention of cell death by addition of caspase inhibitors. Similar results were observed with NFS-70 pro-B cells and select L1210 subclones. In contrast, L1210 parental and L1210 subclone 7-15.6 cells undergo only low levels of apoptosis (< or = 5%). Apoptosis occurred only when DNA (plasmids or salmon sperm DNA) was present during electroporation, but was not dependent on the conformation of the DNA used or the expression of transgenes. Cells pulsed in the presence of dextran sulfate (MW 500,000) did not apoptose. Similar results were observed when L1210 subclone 3-3 was transfected using the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane, although the transfection efficiency and corresponding rate of apoptosis were significantly lower. Applying the caspase inhibitor fluoromethyl ketone (Boc-ASP-FMK) dramatically improved cell viability and transgene expression of select L1210 subclones and NFS-70 pro-B cells.     

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.     

Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.     

Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons

Numerous methods exist for transfecting postmitotic neurons, for example, DNA/calcium phosphate coprecipitation, cationic lipids, viruses, and physical methods such as microinjection, electroporation, and biolistics. Most methods, however, are either toxic to the cell, yield only poor transfection efficiencies, or cells have to be electroporated before plating. In this article, we present a standardized and fast transfection method using DNA/calcium phosphate coprecipitates that efficiently transfer DNA into mature, postmitotic hippocampal neurons. Shifting to CO(2)-independent media with a well-defined pH allows for the tight control of the coprecipitate formation and for adjusting the transfection parameters for the individual DNA plasmid used. The two critical parameters for reproducible and efficient transfections are: the precise pH during crystal formation, and the incubation time of the cells with the coprecipitate. This improved procedure now enables biochemical approaches. By transfecting a dominant-positive Ras mutant, we activate the Erk/MAP kinase signal transduction pathway. Furthermore, using a siRNA plasmid directed against MAP2, the level of an endogenously expressed protein is down-regulated upon transfection. These two approaches demonstrate that the presented transient transfection method can now be used to address questions on a biochemical level in hippocampal neurons.     

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.     

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.     

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.     

人外周血单核细胞的分离和电穿孔法转染

目的:建立分离纯化人外周血单核细胞及转染的有效方法。方法:应用基于HISTOPAQUE的密度梯度离心及抗体标记纯化的方法分离单核细胞,并以电穿孔技术转染绿色荧光蛋白(GFP)表达质粒、肿瘤坏死因子受体相关因子6(TRAF6)小干扰RNA。结果:基于外周血单核细胞的表面标志分子CD14的流式细胞分析表明得到了高纯度(89.95%)的外周血单核细胞;GFP荧光照片显示GFP表达质粒的转染效率为60%～70%;免疫印迹显示TRAF6的表达抑制(90%)下调了脂多糖(LPS)对JNK的激活,说明通过电穿孔技术有效递送了TRAF6小干扰RNA,表明单核细胞若缺少TRAF6将抑制LPS-TLR4信号通路的激活。结论:建立了一种高效的从人外周血中分离原代单核细胞的方法,且应用基于NucleoFectorⅡ的电穿孔技术实现了对此原代单核细胞的高效转染,为进一步外周血单核细胞的相关功能研究奠定了方法学基础。     

Electroporation-mediated transformation of Aeromonas hydrophila

A strain of Aeromonas hydrophila producing copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate, abbreviated as PHBHHx, was successfully transformed by electroporation. The plasmid used was a broad host range plasmid pBBR1MCS. Electroporation conditions were varied systemically to develop an electroporation protocol. The optimal yield of transformant was approximately 4x10(2) CFU/microg DNA at 12.5 kV/cm and 1000 Omega, resulting in a time constant of approximately 5 ms. The A. hydrophila transformants expressed plasmid-encoded resistance to chloromphenicol. Plasmid DNA in the A. hydrophila transformant was stably maintained. This is the first report of transformation of bacteria A. hydrophila.     

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene beta-gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the beta-gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiently using the in situ method. Furthermore, we have measured the time required to express the beta-gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing beta-gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.     

Lipofection and nucleofection of substrate plasmid can generate widely different readings of DNA end-joining efficiency in different cell lines

In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ) – a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters. To help standardize these assays, we searched for parameters underpinning these variations and identified transfection method as an important determinant. Here, we compare a lipid-based transfection method, lipofection, with an electroporation method, nucleofection, and find large, unanticipated and cell line-dependent differences in percent end-joining without recognizable trends. For example, in rodent cells, transfection using lipofection gives nearly WT end-joining in DNA-PKcs mutants and only mildly inhibited end-joining in Lig4 and Ku mutants. In contrast, transfection using nucleofection shows marked end-joining inhibition in all NHEJ mutants tested as compared to the WT. In human HCT116 cells, end-joining after nucleofection is strongly suppressed even in the WT and the differences to the mutants are small. After lipofection, in contrast, end-joining is high in WT cells and markedly suppressed in the mutants. We conclude that better understanding and control of the physicochemical/biological and analytical parameters underpinning these differences will be required to generate with plasmid assays results with quantitative power comparable to that of well-established methods of DSB analysis such as pulsed-field gel electrophoresis or γ-H2AX foci scoring. Until then, caution is needed in the interpretation of the results obtained – particularly with reference to pathway efficiency and residual damage – and confirmation of critical results with alternative transfection approaches is advisable.     

Introduction of the human growth hormone gene into the guinea pig mammary gland by in vivo transfection promotes sustained expression of human growth hormone in the milk throughout lactation

We tested the feasibility of transfecting mammary tissue in vivo with an expression plasmid encoding the human growth hormone (hGH) gene, under the control of the cytomegalovirus promoter. Guinea pig mammary glands were transfected with plasmid DNA infused through the nipple canal and expression was monitored in control and transfected glands by radioimmunoassay of milk samples for hGH. Sustained expression of hGH throughout lactation was attained with a polyion transfection complex shown to be optimal for the transfection of bovine mammary cells, in vitro. However, contrary to expectations, hGH expression was consistently 5- to 10-fold higher when DEAE-dextran was used alone for transfection. Thus polyion complexes which are optimal for the transfection of cells in vitro may not be optimal in vivo. The highest concentrations of hGH in milk were obtained when glands were transfected within 3 days before parturition. This method may have application for studying the biological role or physical properties of recombinant proteins expressed in low quantities, or for investigating the regulation of gene promoters without the need to construct viral vectors or produce transgenic animals.