Calcium Phosphate-mediated Transfection Research Articles

Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Calcium Phosphate-Mediated Transfection of Eukaryotic Cells with Plasmid DNAs.

This protocol describes a calcium phosphate-mediated transfection method for use with plasmid DNAs and adherent cells. At the end of the protocol is an alternative method for high-efficiency generation of stable transfectants.

Calcium Phosphate-Mediated Transfection of Cells with High-Molecular-Weight Genomic DNA.

This protocol describes a calcium phosphate-mediated transfection method for use with high-molecular-weight genomic DNAs.

Calcium Phosphate-Mediated Transfection of Adherent Cells or Cells Growing in Suspension: Variations on the Basic Method.

This protocol describes two variations of the calcium phosphate-mediated transfection method. The first can be used with all types of adherent cells, but is particularly useful for polarized epithelial cells, which do not efficiently take up material by endocytosis through the apical plasma membrane. To improve transfection efficiency, adherent cells are trypsinized and collected by centrifugation. The cells are resuspended in the calcium phosphate-DNA coprecipitate and then plated again on tissue culture dishes. Most lines of cells grown in suspension are resistant to calcium phosphate-mediated transfection methods; however, a few cell lines grown as suspension cultures (e.g., HeLa cells) can be transfected using the second modified calcium phosphate procedure described here.

Identification of Functionally Relevant microRNAs in the Regulation of Allergic Inflammation.

Transgenic methods to manipulate CD4 T lymphocytes in vivo via forced expression of TCR transgenes and targeted "knockout" of individual genes by Cre-lox technology are fundamental to modern immunology. However, efforts to scale up functional analysis by modifying expression of larger numbers of genes in T cells ex vivo have proven surprisingly difficult. Early RNA interference experiments achieved successful small RNA transfection by using very high concentrations of short-interfering RNA (siRNA) [1], but primary T cells are generally resistant to standard electroporation, cationic liposome-, and calcium phosphate-mediated transfection methods. Moreover, although viral vectors can successfully introduce DNA fragments of varying length, expression of these constructs in primary T cells is low efficiency and the subcloning process laborious. In this context, the relatively recent discovery of dozens of highly expressed microRNAs (miRNAs) in the immune system provides both an opportunity and a new challenge [2, 3]. How can we query the miRNAome of a cell to assign particular roles to individual miRNAs? Here, we describe an optimized technique for efficient and reproducible transfection of primary mouse CD4 T cells in vitro with synthetic miRNA mimics.

RNAi Screening of Leukemia Cells Using Electroporation.

RNAi-mediated screening has been an integral tool for biological discovery for the past 15 years. A variety of approaches have been employed for implementation of this technique, including pooled, depletion/enrichment screening with lentiviral shRNAs, and segregated screening of panels of individual siRNAs. The latter approach of siRNA panel screening requires efficient methods for transfection of siRNAs into the target cells. In the case of suspension leukemia cell lines and primary cells, many of the conventional transfection techniques using liposomal or calcium phosphate-mediated transfection provide very low efficiency. In this case, electroporation is the only transfection technique offering high efficiency transfection of siRNAs into the target leukemia cells. Here, we describe methods for optimization and implementation of siRNA electroporation into leukemia cell lines and primary patient specimens, and we further offer suggested electroporation settings for some commonly used leukemia cell lines.

A Conserved Aspartate Residue Located at the Extracellular End of the Binding Pocket Controls Cation Interactions in Brain Glutamate Transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Hetero-oligomerization of Neuronal Glutamate Transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Evaluation of Gene Promoters for Liver Expression by Hydrodynamic Gene Transfer

Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A. Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1 alpha (EF1alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 x 10(5) HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII (FVIII)-deficient mice were similarly injected with CBA- or EF1alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined. In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1alpha-FVIII or CAG-FVIII plasmids survived after tail clipping. The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.

Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.

Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate-DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.

Noncovalently linked nuclear localization peptides for enhanced calcium phosphate transfection

The generation of cell lines stably expressing recombinant material is a lengthy process and there has thus been much interest in the use of transient expression systems to rapidly produce recombinant material. To achieve this, the DNA of interest must be delivered into the nucleus of the target cell. The mechanisms by which this process occurs are poorly understood and the efficiency of various methods differs widely. Recently, nuclear localization signals (NLSs) have been investigated to target entry of DNA into the nucleus of mammalian cells. We have used NLSs from the SV40 and Tat antigens mixed with our model luciferase reporter gene plasmid for the transfection of Chinese hamster ovary (CHO) cells using calcium phosphate and FuGENE 6 transfection technology. The noncovalent complexation of NLSs with plasmid DNA before calcium phosphate-mediated transfection resulted in enhanced reporter gene expression with increasing ratios of NLS to plasmid until reaching a maximum. At higher ratios than maximum expression, the expression levels decreased. On the other hand, when using FuGENE 6 reagent NLSs did not enhance reporter gene expression. Cell cycle arrest in G(2)/M phase obliterated the effect of the NLS on reporter gene expression when using the calcium phosphate transfection method.

Domain Interactions of the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) forms oligomeric structures important for optimal function in binding and internalization of Man-6-P-bearing extracellular ligands as well as lysosomal biogenesis and growth regulation. However, neither the mechanism of inter-receptor interaction nor the dimerization domain has yet been identified. We hypothesized that areas near the ligand binding domains of the receptor would contribute preferentially to oligomerization. Two panels of minireceptors were constructed that involved truncations of either the N- or C-terminal regions of the M6P/IGF2R encompassing deletions of various ligand binding domains. alpha-FLAG or alpha-Myc-based immunoprecipitation assays showed that all of the minireceptors tested were able to associate with a full-length, Myc-tagged M6P/IGF2R (WT-M). In the alpha-FLAG but not alpha-Myc immunoprecipitation assays, the degree of association of a series of C-terminally truncated minireceptors with WT-M showed a positive trend with length of the minireceptor. In contrast, length did not seem to affect the association of the N-terminally truncated minireceptors with WT-M, except that the 12th extracytoplasmic repeat appeared exceptionally important in dimerization in the alpha-FLAG assays. The presence of mutations in the ligand-binding sites of the minireceptors had no effect on their ability to associate with WT-M. Thus, association within the heterodimers was not dependent on the presence of functional ligand binding domains. Heterodimers formed between WT-M and the minireceptors demonstrated high affinity IGF-II and Man-6-P-ligand binding, suggesting a functional association. We conclude that there is no finite M6P/IGF2R dimerization domain, but rather that interactions between dimer partners occur all along the extracytoplasmic region of the receptor.

Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein.

Introduction of recombinant genes in the genome of primary lymphocytes by virtue of a replication-deficient retrovirus can be used in immunological studies and for cell-based gene therapy. Packaging cells GP+E86 producing replication-deficient retrovirus incorporating the genes of enhanced green fluorescent protein (eGFP), C2gamma or C2xi, were generated by calcium phosphate-mediated transfection. Clones with the highest titres of retrovirus vector were isolated from them and their supernatants were used for transduction of PT67 cells. Primary mouse lymphocytes and T-cell hybridoma MD.45 were transduced by centrifugation with retroviral stock. The retroviral content of packaging cell supernatants was determined by dot blotting and hybridization with a DNA probe. PT67 cells produced approximately 50 times more retrovirus vector than the original GP+E86 clones. When retroviral stocks of PT67 and GP+E86 cells were used at 1/50 dilution and undiluted, respectively (to normalize them for retroviral RNA content), the transduction efficiency of mouse T-cell hybridoma was 40% and 5%, respectively. Centrifugation of target cells with retroviral stock at 2000 g for 60 min increased the percentage of transduced cells two- to three-fold. Within a population of cells isolated from the draining lymph nodes of an immunized mouse and reactivated with an antigen, up to 60% of CD4(+) T cells and up to 80% of B cells could be transduced with a transgene in replication-deficient retrovirus packaged by PT67 cells using the optimized gene transfer protocol. This protocol allows for the generation of packaging cells producing high titres of retrovirus vector. The 10A1 envelope protein is superior to the ecotropic one for the transduction of mouse lymphocytes.

Reproducible high efficiency gene transfer into Y79 retinoblastoma cells using adenofection

Several photoreceptor-specific genes are actively transcribed in Y79 retinoblastoma (Rb) cells, making this cell line potentially useful for the study of photoreceptor metabolism. The utility of these cells is limited because commonly used methods of gene transfer into Y79 cells are inefficient and lack reproducibility. In contrast, we found that adenovirus transduction yields high efficiency gene transfer, however, generation of recombinant adenovirus is lengthy and time consuming. Here, we show that adenofection, a method of coupling adenovirus to plasmid DNA for improved gene transfer, is efficient for gene delivery into Y79 cells. Recombinant adenovirus expressing bacterial lacZ was noncovalently complexed to GFP or luciferase reporter plasmids with polyethylenimine. Efficiency of plasmid gene delivery was determined by monitoring GFP fluorescence. For comparison, calcium phosphate-mediated or cationic lipid transfection was performed in Y79 and HEK293 cells using standard protocols. The adenofection protocol yielded significantly higher efficiencies in Y79 cells than that obtained in these cells with calcium phosphate or cationic lipids. This method will facilitate any experiment requiring reproducible high-level gene transfer. Here, we show that adenofection can be used to analyze activity of the rod photoreceptor PDE6A gene promoter.

Transient transfection and adrenergic receptor promoter analysis.

AbstractThere are several methods currently available for transfection of DNA into mammalian cells. These include transfection with calcium phosphate (1), diethylaminoethyl (DEAE)-dextran, cationic liposomes, nonliposomal lipid compounds, and electroporation (2). Each of these methods has advantages and disadvantages; selection of transfection method is based on the cell types used and personal preference. Transfection using calcium phosphate is highly efficient and is the most cost-effective method; therefore, this is the method of choice for larger-scale experiments. It is also the most widely utilized method and has been successful for DNA transfection into a variety of cell types. Our laboratory has used calcium phosphate-mediated transfection to introduce β1-adrenergic receptor (β1-AR) gene fragments into SK-N-MC, C6, and W1 cells (3,4). The protocol for transient transfection of adherent cells with calcium phosphate will be described in this chapter. As with other transfection methods, the efficiency of calcium phosphate-mediated transfection is largely determined by the recipient cell type. An optimized protocol for one cell type does not guarantee success in another cell type. From our experience, lipofectin transfection reagent works well for transfection of β1-AR gene fragments into adherent cell lines, but is much less effective for transfection of DNA into primary cultures. As noted by other investigators (5), we have applied FuGENE™ 6 transfection reagent, a nonliposomal lipid compound, to transfect β1-AR gene fragments into 3-d-old neonatal cardiac myocytes with great efficiency (Fig. 1). The transfection efficiency of two different transfection reagents, FuGENE 6 (Boehringer Mannheim, Indianapolis, IN) and Lipofectin (Gibco-BRL, Grand Island, NY). Cardiac myocytes were prepared from 3-d-old neonatal rats. Cells were transfected with pGL3-control vector 2 d after plating in 24-well plates to test efficiency on primary culture. The DNA doses are indicated. Several doses of Lipofectin reagent were used, ranging from 0.8 to 1.5 μL/well. The ratio of DNA (μg)/FuGENE 6 reagent (μL) was 1/5. Cells were exposed to the transfection reagent for 20 h before harvesting for luciferase activity. Data are mean ± SEM of luciferase relative light unit from four separate experiments. KeywordsTransfection EfficiencyCalcium PhosphateTransient TransfectionCationic LiposomeTransfection MethodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems

This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate-mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion-containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be "pseudotyped" with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV-G-pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.