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The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor transfection efficiency of conventional methods in T cells, there has been an effort to increase the transfection rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery.
Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production.
Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.
Amyloid fibril deposits are a main source of pathology in neurodegenerative diseases. Normal proteins such as tau, alpha-synuclein, TDP-43 and others could form specific conformational fibrils called amyloid, which deposited in the brains of neurodegenerative diseases. Although the pathological roles of amyloids in cell death have been discussed a lot, their other functions have not been investigated well. Here, we studied the effect of amyloids on DNA transfection in vivo. We injected quantum dot labeled or non-labeled amyloid-preformed fibrils (PFFs) and a green fluorescent protein (EGFP) expression vector into organs including brain, testis, liver and calf muscle. GFP expression patterns were examined by immunohistochemistry and western blotting. At 24 h after injection, EGFP was predominantly expressed in the neurons in the cortex and the striatum, Leydig cells in testis, hepatocytes in the liver and muscle cells. EGFP expression was inhibited by an endocytosis inhibitor, sertraline in the brain and testis. The amyloid-PFFs potentiated Ca2+ transients shown by calcium imaging and EGFP expression in the brain was blocked by Ca blocker, cilnidipine. Our results show that amyloid-PFFs facilitate DNA transfection and can be used for a new gene delivery system in vivo.
Despite the wide variety of available cationic lipid platforms for the delivery of nucleic acids into cells, the optimization of their composition has not lost its relevance. The purpose of this work was to develop multi-component cationic lipid nanoparticles (LNPs) with or without a hydrophobic core from natural lipids in order to evaluate the efficiency of LNPs with the widely used cationic lipoid DOTAP (1,2-dioleoyloxy-3-[trimethylammonium]-propane) and the previously unstudied oleoylcholine (Ol-Ch), as well as the ability of LNPs containing GM3 gangliosides to transfect cells with mRNA and siRNA. LNPs containing cationic lipids, phospholipids and cholesterol, and surfactants were prepared according to a three-stage procedure. The average size of the resulting LNPs was 176 nm (PDI 0.18). LNPs with DOTAP mesylate were more effective than those with Ol-Ch. Core LNPs demonstrated low transfection activity compared with bilayer LNPs. The type of phospholipid in LNPs was significant for the transfection of MDA-MB-231 and SW 620 cancer cells but not HEK 293T cells. LNPs with GM3 gangliosides were the most efficient for the delivery of mRNA to MDA-MB-231 cells and siRNA to SW620 cells. Thus, we developed a new lipid platform for the efficient delivery of RNA of various sizes to mammalian cells.
Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca(2+)-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells.
The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.
Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a laser, to produce temporary mechanical deformations of the cell membrane and facilitate the delivery of plasmid DNA into cells. We show that high stress gradients, produced when picosecond laser pulses with a fluence of 100 mJ/cm2 are absorbed by piezophotonic materials, enable transfection of a plasmid DNA encoding Green Fluorescent Protein (gWizGFP, 3.74 MDa) in COS-7 monkey fibroblast cells with an efficiency of 5% at 20 °C, in 10 minutes. We did not observe significant cytotoxicity under these conditions. Photoacoustic transfection is scalable, affordable, enables nuclear localization and the dosage is easily controlled by the laser parameters.
The efficient transfection of functional proteins into cells can serve as a means for regulating cellular processes toward solving fundamental challenges in biology and medicine. However, the use of proteins as effective intracellular agents is hindered by their low cellular uptake and susceptibility to degradation. Over the past 15 years, our group has been developing spherical nucleic acids (SNAs), nanoparticles functionalized with a dense radially oriented shell of nucleic acids. These structures actively enter cells and have opened new frontiers in chemical sensing, biodiagnostics and therapeutics. Recently, we have shown that proteins can be used as structurally precise and homogeneous nanoparticle cores in SNAs. The resultant protein SNAs (ProSNAs) allow previously cell-impermeable proteins to actively enter cells, exhibit high degrees of stability and activity both in cell culture and in vivo, and show enhanced pharmacokinetics. Consequently, these modular structures constitute a plug-and-play platform in which the protein core and nucleic acid shell can be independently varied to achieve a desired function. Here, we describe the synthesis of ProSNAs through the chemical modification of solvent-accessible surface residues (3-5 d). We also discuss design considerations, strategies for characterization, and applications of ProSNAs in cellular transfection, biological sensing and functional enzyme delivery in vivo.
Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to explore the bionano interactions between GOCL/DNA complexes (hereafter referred to as "grapholipoplexes") and human plasma. When exposed to increasing protein concentrations, grapholipoplexes get covered by a protein corona that evolves with protein concentration, leading to biocoronated complexes with modified physicochemical properties. Here, we show that the formation of a protein corona dramatically changes the interactions of grapholipoplexes with four cancer cell lines: two breast cancer cell lines (MDA-MB and MCF-7 cells), a malignant glioma cell line (U-87 MG) and an epithelial colorectal adenocarcinoma cell line (CACO-2). Luciferase assay clearly indicates a monotonous reduction of the transfection efficiency of biocoronated grapholipoplexes as a function of protein concentration. Finally, we report evidence that a protein corona formed at high protein concentrations (as those present in in vivo studies) promotes a higher capture of biocoronated grapholipoplexes within degradative intracellular compartments (e.g., lysosomes), with respect to their pristine counterparts. On the other hand, coronas formed at low protein concentrations (human plasma = 2.5%) lead to high transfection efficiency with no appreciable cytotoxicity. We conclude with a critical assessment of relevant perspectives for the development of novel biocoronated gene delivery systems.
Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.
Schwann cells line nerve fibers in the peripheral nervous system (PNS) and synthesize myelin. In addition, they support neuronal survival, neurite growth and regeneration. In dissociated cultures of postnatal mouse spiral ganglia, regenerating neurites spontaneously associate with Schwann cells. However, the mechanisms and consequences of interactions between cochlear Schwann cells and spiral ganglion neurites have not been examined. Further, the similarities and differences between cochlear Schwann cells and other PNS Schwann cells have not been studied. Experiments to examine these questions will rely on the ability to purify and characterize cochlear Schwann cells. Here we present methods for purifying Schwann cells from postnatal mouse cochleas and for transfecting them with expression plasmids. Dissociated spiral ganglia were plated on poly-D-lysine/laminin in medium containing neurotrophins, leukemia inhibitory factor (LIF), N2 supplement and serum and maintained for 5 days. Cells were harvested with trypsin/EDTA and subjected to an immuno-magnetic purification procedure. After 24 h in vitro, cultures were >85% Schwann cells. Nucleofection of purified Schwann cells with pMax-green fluorescent protein (pMax-GFP) plasmid, or with pEGFP-C-vimentin plasmid returned >45% transfection efficiency. These methods will allow the in-depth characterization of cochlear Schwann cells and an evaluation of their biochemical, functional, and genetic mechanisms that may promote neurite growth from the spiral ganglion.
Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.
We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.
We have characterized a broad range of liposome formulations with varying DcChol:DOPE ratio. Subsequent addition of DcChol to liposomes increases its positive surface charge. However, loading the nuclear acids did not neutralize the overall negative surface potential to a similar extent. The liposomes were tested by transfection of DNA in living cancer cells.
Non-viral vectors for the transfection of genetic material are at the frontier of medical science. In this article, we introduce for the first time, cyclopropenium-containing nanoparticles as a cationic carrier for gene transfection, as an alternative to the common quaternary ammonium transfection agents. Cyclopropenium-based cationic nanoparticles were prepared by crosslinking poly(ethylene imine) (PEI) with tetrachlorocyclopropene. These nanoparticles were electrostatically complexed with plasmid DNA into nanoparticles (~50 nm). Their cellular uptake into F929 mouse fibroblast cells, and their eventual expression in vitro have been described. Transfection is enhanced relative to PEI with minimal toxicity. These cyclopropenium nanoparticles possess efficient gene transfection capabilities with minimal cytotoxicity, which makes them novel and promising candidates for gene therapy.
Monoclonal antibodies have a variety of applications in research and medicine. Here, we report development of a new method for production of monoclonal antibodies. Our method relies on in vivo RNA transfection rather than peptide vaccination. We took advantage of RNA transcripts complexed with DOTMA and DOPE lipids to transfect mice. Intravenous administration of our RNA vaccine to mice resulted in expression of the antigenic peptides by splenic dendritic cells and detection of the antigens in the serum. The RNA vaccine stimulated production of specific antibodies against the RNA-encoded peptides. We produced monoclonal antibodies against viral, bacterial, and human antigens. In addition, we showed that our RNA vaccine stimulated humoral immunity and rescued mice infected with influenza A virus. Our method could be used as an efficient tool to generate monoclonal antibodies and to stimulate humoral immunity for research and medical purposes.
Transfection is a powerful tool that enables introducing foreign nucleic acids into living cells in order to study the function of a gene product. Ever since the discovery of transfection many side effects or artifacts caused by transfection reagents have been reported. Here, we show that the transfection reagent, JetPRIME alters the localization of the splicing protein SC35 widely used as a nuclear speckle marker. We demonstrate that transfection of plasmids with JetPRIME leads to enlarged SC35 speckles and SC35 cytoplasmic granules. By contrast, transfection of the same plasmid with Lipofectamine 3000 does not have any effect on SC35 localization. The formation of SC35 cytoplasmic granules by JetPRIME-mediated transfection is independent of exogenous expression by plasmid and although similar in morphology they are distinct from P-bodies and stress granules. This method of transfection affected only SC35 and phosphorylated SR proteins but not the nuclear speckles. The JetPRIME-mediated transfection also showed compromised transcription in cells with enlarged SC35 speckles. Our work indicates that the use of JetPRIME alters SC35 localization and can affect gene expression and alternative splicing. Therefore, caution should be exercised when interpreting results after the use of a transient transfection system, particularly when the subject of the study is the function of a protein in the control of gene expression or mRNA splicing.
Stable transfection systems have been described in many protozoan parasites, including Plasmodium falciparum, Cryptosporidium parvum, Babesia bovis, Babesia ovata, and Babesia gibsoni. For Babesia sp. Xinjiang (Bxj), which is the causative pathogen of ovine babesiosis and mainly prevails across China, the platform of those techniques remains absent. Genetic manipulation techniques are powerful tools to enhance our knowledge on parasite biology, which may provide potential drug targets and diagnostic markers.
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