Comparison of lipid-mediated and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.     

Optimizing the generation of stable neuronal cell lines via pre-transfection restriction enzyme digestion of plasmid DNA

Transfection of mammalian cell lines is a widely used technique that requires significant optimization, including transfection method or product used, DNA vector, cell density, media composition and incubation time. Generation and isolation of stable transfectants from the large pool of untransfected or only transiently transfected cells can be laborious and time-consuming. Transfection of DNA is usually performed with a non-linearized plasmid, since it is assumed that cutting the plasmid beforehand leads to a lower efficiency of transfection or the degradation of linearized DNA by cytosolic nucleases. However, the transfected circular plasmid will be linearized by a random cut within the cell and it might be possible that sensitive parts of the plasmid such as the resistance gene or the gene of interest are destroyed upon linearization. On the other hand, linearizing a plasmid before transfection by a single, defined cut with a selected restriction enzyme in a non-coding area of the gene has the advantage of ensuring the integrity of all necessary gene elements of the plasmid. In this study, we have compared these different methods in order to increase both transient and stable transfection efficiency in mammalian cells. We report that linearization of plasmid DNA prior to transfection can increase both the efficiency of stable clone generation and target gene expression, but is dependant on the site of linearization within the vector.     

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

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.     

Salt ions and related parameters affect PEI–DNA particle size and transfection efficiency in Chinese hamster ovary cells

Transfection efficiency is directly associated with the expression level and quantity of recombinant protein after the transient transfection of animal cells. The transfection process can be influenced by many still-unknown factors, so it is valuable to study the precise mechanism and explore these factors in gene delivery. Polyethylenimine (PEI) is considered to have high transfection efficiency and endosome-disrupting capacity. Here we aimed to investigate optimal conditions for transfection efficiency by setting different parameters, including salt ion concentration, DNA/PEI ratio, and incubation time. We examined the PEI–DNA particle size using a Malvern particle size analyzer and assessed the transfection efficiency using flow cytometry in Chinese hamster ovary-S cells. Salt ions, higher amounts of PEI tended to improve the aggregation of PEI–DNA particles and the particle size of PEI–DNA complexes and the transfection efficiency were increased. Besides, the particle size was also found to benefit from longer incubation time. However, the transfection efficiency increased to maximum of 68.92 % at an incubation time of 10 min, but decreased significantly thereafter to 23.71 %, when incubating for 120 min (P < 0.05). Besides, PEI–DNA complexes formed in salt-free condition were unstable. Our results suggest DNA and PEI incubated in 300 mM NaCl at a ratio of 1:4 for 10 min could achieve the optimal transfection efficiency. Our results might provide guidance for the optimization of transfection efficiency and the industrial production of recombinant proteins.     

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines. Revisions requested 27 October 2005; Revisions received 12 December 2005     

Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes

The application of conventional cationic liposomes/DNA complexes in gene transfer was hampered due to their large size, instability, and limited transfection site in vivo. In this report, we described a dialysis-based method and produced small, stable, and negatively charged DNA-containing liposomes composed of low content of cationic lipid and high content of fusogenic lipid. The liposomes were relatively spherical with a condensed core inside, and exhibited small size with narrow particle size distribution. The encapsulation efficiency of the liposomes was 42.53 +/- 2.29%. They were stable and showed enough protective ability to plasmid DNA from degradation after incubation with different amounts of DNase. Twenty-fold higher transfection efficiency for the liposomes was achieved when compared with that of naked plasmid DNA and no toxicities to hepatocellular carcinoma cells were observed. Our results indicate that the negatively charged DNA-containing liposomes can facilitate gene transfer in cultured cells, and may alleviate the drawbacks of the conventional cationic liposomes/DNA complexes for gene delivery in vivo.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes

We have previously shown that pH-sensitive immunoliposomes can mediate a target-specific delivery of plasmid DNA to tumor cells grown in a mouse model [Wang, C.-Y., & Huang, L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7851-7855]. The efficiency of delivery in terms of the target cell transformation frequency has now been characterized for both short- and long-term gene expression in a tissue culture system. Herpes simplex virus thymidine kinase (TK) gene was used as a reporter gene. It was placed under the control of the promoter for the rat phosphoenolpyruvate carboxykinase gene, which contains a cAMP regulatory element. Therefore, the expression of the exogenous gene in the target cell, mouse Ltk- cells, can be regulated by cAMP drugs. The plasmid DNA was encapsulated in liposomes using a detergent dialysis method. The efficiency of gene delivery was optimized with respect to the time course and dose of liposome-associated DNA. The existence of antibody of the liposomes was essential for the maximal level of DNA delivery. Delivery was also dependent on the lipid composition of the liposome. The pH-sensitive lipid composition gave 8-fold higher efficiency than the corresponding pH-insensitive composition. The transformation efficiency of the target cell also depended on the regulation of gene expression; cells incubated with dibutyryl-cAMP and theophylline showed a much higher level of transformation frequency than cells incubated without the drugs. When all liposome and incubation parameters are optimized, the Ltk- cells showed a 47% efficiency for the short-term transformation, and 2% for the long-term transformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

DNA-mediated gene transfer as an indicator of DNA damage and its repair by recipient cells

Preparations of plasmid containing the thymidine kinase gene (pHSV106) were treated with the alkylating agents methyl methanesulphonate or N-methyl-N-nitrosourea prior to transfection into thymidine kinase-deficient mouse L-cells using the DNA-calcium phosphate co-precipitation technique. Relative to transfection with unmodified plasmid, a reduced transformation efficiency was observed using alkylation-damaged plasmid, N-methyl-N-nitrosourea causing the greatest inhibition. Treatment of recipient cells with arabinosyl cytosine or dideoxythymidine during the expression period following transfection by the 'damaged' plasmid reduced transformation efficiency, suggesting that DNA repair 4-6 h post-transfection was required for gene expression.     

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways.     

Enhancement and optimization of plasmid expression in femtosecond optical transfection

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non‐invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3‐fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO‐K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ～70% without compromising the transfection efficiency. Also, we report for the first time the successful photo‐transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.     

High-frequency transfection of mouse FM3A mammary carcinoma cells in suspension culture with plasmid DNA

High-frequency transfection of mouse FM3A cells, grown in suspension, with plasmid pSV2neo DNA was achieved by incubation of the cells with DNA plus polybrene for 6 h followed by an osmotic shock with a hypertonic NaCl solution. When incubated for 20 min at 34 degrees C, FM3A cells showed resistance to the osmolarity change from 0.1 to 9.0% NaCl in the medium. Within this concentration range, 5-7% gave the highest efficiency of transfection. Both linear and circular forms of plasmid DNA produced transformants with equal efficiency. This method was simple, reproducible, and carrier DNA was not required. The efficiency was about 100 times higher than that of the method with DNA-calcium phosphate precipitates. Transformed cells were stable and different numbers of plasmid DNA copies were detected.     

Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen

Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal‐cell biotechnology. Difficult‐to‐express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full‐length protein constructs. Post‐translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time‐, labor‐, and cost‐intensive. It is clearly desirable to find an automated and miniaturized fast multi‐sample screening method for protein expression in such systems. With this in mind, in this paper a high‐throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide‐binding Oligomerization Domain‐containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid‐based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. Biotechnol. Bioeng. 2016;113: 1975–1983. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Generation of recombinant baculovirus via liposome-mediated transfection

Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of transfecting insect cells exist, we have found liposome-mediated transfection to be highly efficient. Here we detail the protocols and medium needed for efficient, simple transfection of Spodoptera frugiperda cells.