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.     

High-efficiency transfection of mammalian neurons via nucleofection

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.     

Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90-95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.     

Investigation and circumvention of transfection inhibition by ferric ammonium citrate in serum-free media for Chinese hamster ovary cells

While reliable transfection methods are essential for Chinese hamster ovary (CHO) cell line engineering, reduced transfection efficiencies have been observed in several commercially prepared media. In this study, we aimed to assess common media additives that impede efficiency mediated by three chemical transfection agents: liposomal-based (Lipofectamine 2000), polymer-based (TransIT-X2), and lipopolyplex-based (TransIT-PRO). An in-house GFP-expressing vector and serum-free medium (BCR-F12: developed for the purposes of this study) were used to analyze transient transfection efficiencies of three CHO cell lines (CHO-K1, DG44, DP12). Compared to a selection of commercially available media, BCR-F12 displayed challenges associated with transfection in vendor-prepared formulations, with no detection when liposomal-based methods were used, reduced (<3%) efficiency observed when polymer-based methods were used and only limited efficiency (25%) with lipopolyplexes. Following a stepwise removal protocol, ferric ammonium citrate (FAC) was identified as the critical factor impeding transfection, with transfection enabled with the liposomal- and polymer-based methods and a 1.3- to 7-fold increased lipopolyplex efficiency observed in all cell lines in FAC-depleted media (−FAC), although lower viabilities were observed. Subsequent early addition of FAC (0.5–5 hr post-transfection) revealed 0.5 hr post-transfection as the optimal time to supplement in order to achieve transfection efficiencies similar to −FAC medium while retaining optimal cellular viabilities. In conclusion, FAC was observed to interfere with DNA transfection acting at early stages in all transfection agents and all cell lines studied, and a practical strategy to circumvent this problem is suggested.     

Efficient transfection and expression of heterologous genes in PC12 cells

The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.     

原代大鼠海马神经元的高效转染

用原代大鼠海马神经元为模型,对新型电转染方法Nucleofector^TM与脂质体DOTAP和Lipofectaimine^TM的转染效率和转染前后细胞存活率进行比较研究,探讨Nucleofector^TM的高效性与可靠性。从E18胎鼠海马中取出神经元进行体外培养,并用神经微丝(NF)抗体进行免疫细胞化学染色鉴定细胞类型。分别用DOTAP,Lipofectamine^TM and Nucleofector^TM包裹pCMV-eGFP质粒转染原代大鼠海马神经元。神经元的存活率用流式细胞仪检测。实验结果表明：DOTAP和Lipofectamine^TM的基因转染效率仅为1．55％和2．45％,而Nucleofector^TM的转染效率则超过20％;细胞转染前后的存活率在DOTAP组分别为98．37％和88．35％,Lipofectamine^TM组分别为98．37％和90．11％,而在Nucleofector^TM组中分别为98．37％和51．82％。上述实验数据表明：Nucleofector^TM转染技术能高效并安全地转染原代大鼠海马神经元,但死亡率较高。     

Serum-free large-scale transient transfection of CHO cells

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10     

RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells

The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.     

An electroporation protocol for efficient DNA transfection in PC12 cells

A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.     

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.     

Ultrasound-mediated gene transfer into neuronal cells

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.     

Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons

RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.     

Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells

Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.     

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.     

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types.     

Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.     

Suspended-drop electroporation for high-throughput delivery of biomolecules into cells

We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.     

Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons

RNA interference (RNAi) provides a powerful experimental tool for sequence-specific gene silencing, allowing efficient analysis of gene function in a multitude of cell types. However, application of RNAi in primary mammalian neurons has been limited by low-transfection efficiency and considerable toxicity of conventional transfection methods. In this study, we evaluated a peptide-mediated and a polymer/lipid-based cellular delivery method for siRNA into rat primary neurons and compared the results with a commonly used liposomal transfection reagent. Stearylated octaarginine (Stearyl-R8) was used as polypeptide and artificial virus-like particles (AVPs) were used as a combined liposomal-polymeric vector, since both reagents have been previously shown to successfully transfect DNA into cell lines. Stearyl-R8 and AVPs both promoted siRNA transfection into primary hippocampal neurons via the endosomal pathway. SiRNA-mediated gene silencing could be effectively induced in primary neuron cultures. In comparison with the commonly used cationic liposome transfection agent, both novel reagents were less detrimental to cell metabolic activity. We conclude that these novel transfection methods yield performances comparable to cationic liposome-mediated transfection for siRNA, while being less cytotoxic in primary neurons. Stearyl-R8 and AVPs may therefore represent novel and more cost-efficient alternatives to conventional siRNA-transfection reagents.     

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.