Cationic Lipid Transfection Research Articles

Primary Neuronal Culture and Transient Transfection.

Primary neuronal culture and transient transfection offer a pair of crucial tools for neuroscience research, providing a controlled environment to study the behavior, function, and interactions of neurons in vitro. These cultures can be used to investigate fundamental aspects of neuronal development and plasticity, as well as disease mechanisms. There are numerous methods of transient transfection, such as electroporation, calcium phosphate precipitation, or cationic lipid transfection. In this protocol, we used electroporation for neurons immediately before plating and cationic lipid transfection for neurons that have been cultured for a few days in vitro. In our experience, the transfection efficiency of electroporation can be as high as 30%, and cationic lipid transfection has an efficiency of 1%-2%. While cationic lipid transfection has much lower efficiency than electroporation, it does offer the advantage of a higher expression level. Therefore, these transfection methods are suitable for different stages of neurons and different expression requirements. Key features • Culture of primary neurons from the CNS. • Electroporation for freshly isolated neurons in suspension. • Cationic lipid transfection for adherent neurons.

Cationic lipid transfection induces nuclear actin filaments.

Cationic lipids are widely used for gene delivery. Here, we report the transient formation of nuclear actin filaments in mammalian cells transfected with commercially available transfection reagents regardless of the proteins transfected. Readily detectable with phalloidin, nuclear actin ranges from short filaments to a fully developed network. Nuclear actin filaments persist for hours, peak 20 h after transfection, and may be involved in DNA damage repair.

Producing genetically engineered macrophages with enhanced immunity via microinjection.

Many virus-mediated and chemical-based methods for delivering foreign genes into target cells, such as recombinant lentivirus transfection and cationic lipid transfection, are remarkably challenging to use on immune cells because of low efficiency and high toxicity. Microinjection is a promising method to deliver foreign gene expression plasmids into single macrophages directly. This paper reports a new method that can be used to produce a genetically engineered macrophage cell line with enhanced immunity through a home-made high-throughput microinjection system. Microinjection of the expression plasmid carrying a mouse-derived toll-like receptor 4 (Tlr4) gene into a mouse macrophage cell line (Raw264.7) can construct a new stable cell line overexpressing the target gene. The expression efficiency of the target gene in the injected Raw264.7 cells reached 90%, which was measured by injecting a particular plasmid carrying a fused enhanced green fluorescent protein (eGFP) gene fragment with the Tlr4 gene and counting the proportion of cells that emitted green fluorescence. Further assessment of the messenger RNA (mRNA) and protein produced by the Tlr4 gene indicated that its expression was up-regulated remarkably in successfully injected cells. The expression of downstream genes of Tlr4 in injected cells was higher than in untouched cells. Microinjection can avoid polarization effects, which are common when traditional transfection methods are used. A case study was conducted to verify that the injected macrophages overexpressing Tlr4 could activate downstream signaling pathways and showed enhanced inhibition effect on tumor cell migration and invasion. The success of this research will verify that microinjection can be an efficient and safe method in cell transfection applications.

A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.

BackgroundThe genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants.ResultsHere, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1.ConclusionsOur results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

Tips for Successful lncRNA Knockdown Using Gapmers.

Long noncoding RNAs (lncRNAs) are a recently discovered class of RNA that have diverse intracellular regulatory and structural roles. Because of their wide assortment of functions, lncRNAs can have varied distributions in the nucleus and/or cytoplasm of a cell. However, even though tens of thousands of human lncRNAs have been identified, currently less than 3% have empirically validated functions. RNA knockdown is now a relatively commonplace laboratory technique used to functionally characterize an RNA. These techniques (most commonly antisense therapy and RNA interference) can even have therapeutic benefit to treat a wide variety of genetic or infectious diseases as evidenced by the several RNA knockdown reagents currently in clinical trials. This protocol describes the use of validated gapmer antisense oligonucleotides (ASOs) to knockdown human MALAT1, a nuclear-retained lncRNA that is upregulated in multiple cancer cells. Methods used include cationic lipid transfection into HeLa cells, RNA isolation, and RT-qPCR analysis of the RNA knockdown levels.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost‐efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non‐lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

335. High Content Analysis Platform for Optimization of Lipid Mediated CRISPR-Cas9 Delivery Strategies in Human Cells

Background: Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. Methods: By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of an endogenous and fluorescent mCherry reporter transgene within human embryonic cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. Results: The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression 24 hours after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. After purifying the population for high Cas9 expression, 24 hours after CRISPR-Cas9 delivery, greater than 92% of cells were properly edited. Significance: These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery.View Large Image | Download PowerPoint Slide

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. Statement of SignificanceCRISPR-Cas9 is a new gene-editing technology for “genome surgery” that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type.

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BackgroundDeoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.ResultsIn the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG).Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment.In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups.In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.ConclusionTreatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10.The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0452-3) contains supplementary material, which is available to authorized users.

On the possible involvement of bovine serum albumin precursor in lipofection pathway

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10 percent (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10 percent FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.

Efficient Transfection of DNA into Primarily Cultured Rat Sertoli Cells by Electroporation1

The expression of exogenous DNA in Sertoli cells is essential for studying its functional genomics, pathway analysis, and medical applications. Electroporation is a valuable tool for nucleic acid delivery, even in primarily cultured cells, which are considered difficult to transfect. In this study, we developed an optimized protocol for electroporation-based transfection of Sertoli cells and compared its efficiency with conventional lipofection. Sertoli cells were transfected with pCMV-GFP plasmid by square-wave electroporation under different conditions. After transfection of plasmid into Sertoli cells, enhanced green fluorescent protein (EGFP) expression could be easily detected by fluorescent microscopy, and cell survival was evaluated by dye exclusion assay using Trypan blue. In terms of both cell survival and the percentage expressing EGFP, 250 V was determined to produce the greatest number of transiently transfected cells. Keeping the voltage constant (250 V), relatively high cell survival (76.5% ± 3.4%) and transfection efficiency (30.6% ± 5.6%) were observed with a pulse length of 20 μm. The number of pulses significantly affected cell survival and EGFP expression (P < 0.001). Cell survival clearly decreased following one to three pulses, from 83.9% ± 6.1% to 3.2% ± 1.1%, with EGFP expression increasing from 41.8% ± 9.4% to 66.7% ± 5.2%. The yield of positive cells increased with increasing concentration of plasmid DNA (range, 10-50 μg/ml), from 14.0% ± 2.8% to 35.0% ± 6.3%, but cell viability steadily decreased following 20 μg/ml plasmid DNA, from 73.1% ± 4.9% to 57.0% ± 6.6%. Compared with two popular cationic lipid transfection methods, the transfection efficiency of electroporation (21.5% ± 5.7%) was significantly higher than those of Lipofectamine 2000 (2.9% ± 1.0%) and Effectene (1.9% ± 0.8%) in this experiment (P < 0.001). We describe the process of optimizing electroporation conditions, and the successful electroporation of plasmid DNA into primarily cultured Sertoli cells. Our results indicate that the method of electroporation is more suitable than other approaches for the transfection of Sertoli cells.

Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid (“Umbrella”) and Cholesterol Conjugates Delivered by Cationic Lipids

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC(50) ∼ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.

A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs

A single microRNA (miRNA) can inhibit a large number of mRNA transcripts. This widespread regulatory function has been experimentally demonstrated for a number of miRNAs. However, even when a multitude of targets is confirmed, function of a miRNA is frequently interpreted through a prism of a handful arbitrarily selected “interesting” targets. In this work we first show that hundreds of transcripts with target sites for two miRNAs expressed endogenously in neurons, miR-124 and miR-434-3p, are coordinately upregulated in a variety of neuronal stresses. This creates a landscape where these two miRNAs can exert their widespread inhibitory potential on stress-induced transcripts. Next, we experimentally demonstrate that overexpression of these two miRNAs indeed significantly inhibits expression of hundreds of stress-induced transcripts, thus confirming that these transcripts are enriched in true targets of examined miRNAs. A number of miRNAs were previously shown to have important roles in the regulation of stress responses, and our results suggest that these roles should be understood in light of a wide spread activation of miRNA targets during stresses. Importantly, a popular cationic lipid transfection reagent triggers such induction of miRNA targets. Therefore, when a transfection paradigm is employed to study miRNA function, the results of such studies should be interpreted with consideration for the inadvertent induction of miRNA targets.

Influence of Minor Backbone Structural Variations in Modulating the in Vitro Gene Transfer Efficacies of α-Tocopherol Based Cationic Transfection Lipids

Toward probing the influence of backbone structural variation in cationic lipid mediated gene delivery of α-tocopherol based lipids, two novel α-tocopherol based lipids 1 and 2 have been designed and synthesized. The only structural difference between the cationic amphiphiles 1 and 2 is the backbone structure, where lipid 1 has a non-glycerol backbone and lipid 2 has a glycerol backbone. The lipids 1 and 2 showed contrasting transfection efficiencies: lipid 1 showed high gene transfer efficacy in multiple cultured animals cell lines, whereas lipid 2 is transfection incompetent. In summary, the present findings demonstrate that in the case of α-tocopherol based lipids even minor structural variations like backbone can profoundly influence size, DNA binding characteristics, cellular uptake, and consequently gene delivery efficacies.

Physical–chemical characterization of novel cationic transfection lipids and the binding of model DNA at the air–water interface

Two novel cationic lipids designed for gene transfection were characterized at the air–water interface with respect to their physical–chemical properties and monolayer structures on pH 4 and pH 8 subphases. The binding of model DNA to these monolayers was quantified for the first time by IRRAS, and the structures of the 2D lipoplexes were studied by GIXD and XR. The results have been correlated with 3D structures and with transfection results of former work. The lipids exhibit the same head group structure but differ in their aliphatic chain pattern. While lipid 7 shows strong interactions in the chain and head group region (hydrogen bonds) and forms a highly ordered phase with small cross-sectional areas, the chains of lipid 8 are in the liquid-disordered state on both subphases. The amount of DNA bound to the monolayers of lipids 7 and 8 is comparable at both pH values (considering the same area per molecule) and depends predominantly on the charge density in the monolayer. Surprisingly, even at pH 8 both lipids bind considerable amounts of DNA. Obviously, the approaching macromolecule leads to a further protonation of the lipid monolayer, and in turn more DNA will bind. The interaxial repeat distance of DNA strands is strongly dependent on the phase state of the lipids. While the compression of a liquid-like monolayer (lipid 8) leads to a strong decrease of dDNA, the DNA repeat distances do not change significantly upon compression when adsorbed to a highly charged condensed monolayer (lipid 7).

Nontoxic Genetic Engineering of Mesenchymal Stem Cells Using Serum-Compatible Pullulan-Spermine/DNA Anioplexes

Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier ("anioplex") based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 μg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%-60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain.

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.

A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

Synthesis of cationic lipid transfection agents with O,O- or N,O-acetal linkages

The synthesis of cationic lipids on the basis of 1,2-di-O-tetradecylglycerol is described. The cationic groups represented by the residues of nitrogen-containing heterocyclic bases are attached to the diglyceride backbone through O,O- and N,O-acetal bonds. The resulting lipids can be used as nonviral gene delivery systems to eukaryotic cells.

Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.

BackgroundInterferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ.ResultsTransfection of murine bone-marrow derived macrophages (BMDMs) with a non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response.ConclusionOur results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated with type I and type II IFN signalling and a suppression of macrophage M1 polarization.