Determination of the role of miR-451a on Plasmodium falciparum red blood cell stages, oxidative stress, and proteomic profiling.

BACKGROUND: This study examines the feasibility and effects of introducing microRNA mimic into red blood cells (RBCs) at the initial phases of Plasmodium falciparum 3D7 (Pf3D7) infection. The aim is to determine the correlation between increased expression of miR-451a and parasitaemia. METHODS: In this study miR-mimic-451a labelled with Cy3 and transfected into control and infected RBCs using lipofectamine and analysed using the fluorescence microscopy and flow cytometry. The study demonstrated the efficacy of miR-451a by treating pre-and post-transfected control RBCs and Pf3D7-infected RBCs with miR-mimic-451a. We also examined its impact on % growth inhibition of Pf3D7, oxidative stress markers (Luminometry, LPO, SOD, CAT, GSH and GPx). Additionally, determination of pH, haemoglobin (Hb), and proteomic profile performed using SDS-PAGE. RESULTS: Modified expression level of mir-451a has the potential to change the progression of the infection and yielded a 50% decrease in parasitaemia within 48 h. Moreover, transfected samples were shown to be efficacious in counteracting the oxidative stress-induced alterations during Pf3D7 infection and enable to return the cells towards the normalcy. Modified proteomic profile of transfected iRBCs demonstrates the correlation between overexpression of miRNA and protein expression. where, the major changes were observed in the heavy molecular weight proteins more than 57 kDa. CONCLUSION: The study reveals promising effects of miR-mimic-451a enrichment during RBC stages of Pf3D7, offering insights into potential malaria therapeutic strategies and potential biomedical research implications.

Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

Polyprenol-Based Lipofecting Agents for In Vivo Delivery of Therapeutic DNA to Treat Hypertensive Rats.

Development of efficient vectors for transfection is one of the major challenges in genetic engineering. Previous research demonstrated that cationic derivatives of polyisoprenoids (PTAI) may serve as carriers of nucleic acids. In the present study, the effectiveness of two PTAI-based formulations (PTAI-6-8 and 10-14) was investigated and compared to the commercial reagents. The purpose of applied gene therapy was to enhance the expression of vascular endothelial growth factor (VEGF-A) in the renal medulla of spontaneously hypertensive rats (SHR) and to test its potential as a novel antihypertensive intervention. In the first part of the study (in vitro), we confirmed that PTAI-based lipoplexes efficiently transfect XC rat sarcoma cells and are stable in 37 °C for 7 days. In the in vivo experiments, we administered selected lipoplexes directly to the kidneys of conscious SHR (via osmotic pumps). There were no blood pressure changes and VEGF-A level in renal medulla was significantly higher only for PTAI-10-14-based formulation. In conclusion, despite the promising results, we were not able to achieve VEGF-A expression level high enough to verify VEGF-A gene therapy usefulness in SHR. However, results of our study give important indications for the future development of PTAI-based DNA carriers and kidney-targeted gene delivery.

Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells.

Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Transfection efficiency and cytotoxicity of polyethyleneimine-coated magnetic iron oxide nanoparticles in rooster sperm cells.

Since the morphology of the rooster spermatozoa is different to other animal spermatozoa, the aim of the current study was to investigate the transfection efficiency and cytotoxicity of polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (MION) on these cells. Liposome/nucleic acid (NA) complexes and PEI-coated MION linked to the labeled oligonucleotides were used. Viability and percentage of exogenous nucleic acid uptake of spermatozoa were measured by flow cytometry analyses. The results showed a significant increase in exogenous nucleic acid uptake by rooster spermatozoa (P < 0.001) when treated with MION-NA complexes in comparison to liposome/NA. There were no significant differences between efficiency of lipid-based transfection agent and MION (P > 0.05) during short incubation period. MION with or without magnetic field, did not show significant cytotoxicity while the lipid-based transfection agent significantly decreased (P < 0.05) the viability of rooster spermatozoa. Results of this study showed that magnetofection and lipofection were both effective methods which increased exogenous nucleic acid uptake by rooster spermatozoa. However, the magnetofection method was more successful in maintaining the cell's survival than lipofection method.

Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon).

To achieve in creating permanent shrimp cell lines, cellular arrest of primary cells in the culture is needed to be firstly solved. Considering the insertion of some markers affecting cellular proliferation into primary haemocytes in order to produce the black tiger shrimp cell line and the very low percent of transduced cells previously reported in penaeid shrimps, these paved us the way to set up suitable gene delivery protocols to increase percent of transduced cells in the shrimp as our primary aim. In this study, electroporation and lipofection were used to transfer construct plasmids (pLL3.7 plasmids containing CMV promoters and pGL-IE1-126(A)-EGFP plasmids carrying WSSV IE1 promoters) into primary haemocytes. As it was difficult to distinguish between cells expressing EGFP signal and auto-fluorescence of many dead cells occurred by electroporation during the first 72 h of experiment; so, only lipofection was managed to deliver plasmids into primary cells. Surprisingly, numbers of suspected proliferative cells were derived after electroporation with no insertion of immortalising markers. These cells survived in vitro for up to 45 days with high rate of cell viability, but the number of viable cells decreased throughout the experiment. In addition, these cells expressed genes and proteins closely related to hyaline cells determined using RT-PCR and western blot. For the lipofection experiment, no green fluorescence signal was detected in any primary cell introduced with these plasmids, suggesting that plasmids were not successfully inserted into cells. Also, a number of primary haemocytes had the apoptotic cell death characteristic within 5 days after lipofection. These possibly result from using inappropriate lipofection protocol and chemical substances. In summary, finding out suitable protocols to elevate the percent of transduced cells is still necessary. Additionally, continuous shrimp cell lines would be possibly established by transforming suspected proliferative cells derived from electroporation in this study.

Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P < 0.05) concomitant with the marked inhibition of blastocyst development. Our proposed method, tentatively named 'Octo-lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

Interferon-ß gene transfer inhibits melanoma cells adhesion and migration.

We evaluated the effects of expression of interferon-ß (IFNß) after lipofection on melanoma cells adhesion and migration. Three canine mucosal (Ak, Br and Ol) and one human dermal (SB2) melanomas were assayed. By means of the wound healing assay, we found a significant inhibitory effect of canine IFNß gene expression on cells migration in Br and Ol monolayers. This effect could be reproduced on unlipofected Ol cells with conditioned culture media obtained from canine IFNß gene-lipofected Ol cells, and with recombinant human IFNß on unlipofected SB2 cells. Furthermore, IFNß gene expression of the four tested tumor cells significantly inhibited their adhesion to extracellular matrix (ECM) proteins and their spreading from multicellular spheroids onto gelatin coating. The addition of catalase reverted the increase of reactive oxygen species (ROS) in Ol cells and the inhibition of cell migration in monolayers (Ol) and spheroids (Ol an SB2) produced by canine and human IFNß expression, suggesting the involvement of ROS as mediators of IFNß action on the cells interactions with ECM. Together with its known immune, antiangiogenic and cytotoxic effects, the present data strongly support more studies exploring the clinical potential of IFNß for cancer therapy.

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus.

BACKGROUND: Cationic derivatives of polyprenols (trimethylpolyprenylammonium iodides - PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine. METHODS: Several vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. RESULTS: The mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid. CONCLUSION: The animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Engineering Exosomes for Cancer Therapy.

There remains an urgent need for novel therapeutic strategies to treat metastatic cancer, which results in over 8 million deaths annually worldwide. Following secretion, exosomes are naturally taken up by cells, and capable of the stable transfer of drugs, therapeutic microRNAs and proteins. As knowledge of the biogenesis, release and uptake of exosomes continues to evolve, and thus also has interest in these extracellular vesicles as potential tumor-targeted vehicles for cancer therapy. The ability to engineer exosome content and migratory itinerary holds tremendous promise. Studies to date have employed viral and non-viral methods to engineer the parent cells to secrete modified exosomes, or alternatively, to directly manipulate exosome content following secretion. The majority of studies have demonstrated promising results, with decreased tumor cell invasion, migration and proliferation, along with enhanced immune response, cell death, and sensitivity to chemotherapy observed. The studies outlined in this review highlight the exciting potential for exosomes as therapeutic vehicles for cancer treatment. Successful implementation in the clinical setting will be dependent upon establishment of rigorous standards for exosome manipulation, isolation, and characterisation.

Cytotoxic effects induced by interferon-ω gene lipofection through ROS generation and mitochondrial membrane potential disruption in feline mammary carcinoma cells.

Progress in comparative oncology promises advances in clinical cancer treatments for both companion animals and humans. In this context, feline mammary carcinoma (FMC) cells have been proposed as a suitable model to study human breast cancer. Based on our previous data about the advantages of using type I interferon gene therapy over the respective recombinant DNA derived protein, the present work explored the effects of feline interferon-ω gene (fIFNω) transfer on FMC cells. Three different cell variants derived from a single spontaneous highly aggressive FMC tumor were successfully established and characterized. Lipofection of the fIFNω gene displayed a significant cytotoxic effect on the three cell variants. The extent of the response was proportional to ROS generation, mitochondrial membrane potential disruption and calcium uptake. Moreover, a lower sensitivity to the treatment correlated with a higher malignant phenotype. Our results suggest that fIFNω lipofection could offer an alternative approach in veterinary oncology with equal or superior outcome and with less adverse effects than recombinant fIFNω therapy.

Synthesis and structure-activity relationships of novel cationic lipids with anti-inflammatory and antimicrobial activities.

Certain membrane-active cationic steroids are known to also possess both anti-inflammatory and antimicrobial properties. This combined functionality is particularly relevant for potential therapies of infections associated with elevated tissue damage, for example, cystic fibrosis airway disease, a condition characterized by chronic bacterial infections and ongoing inflammation. In this study, six novel cationic glucocorticoids were synthesized using beclomethasone, budesonide, and flumethasone. Products were either monosubstituted or disubstituted, containing one or two steroidal groups, respectively. In vitro evaluation of biological activities demonstrated dual anti-inflammatory and antimicrobial properties with limited cytotoxicity for all synthesized compounds. Budesonide-derived compounds showed the highest degree of both glucocorticoid and antimicrobial properties within their respective mono- and disubstituted categories. Structure-activity analyses revealed that activity was generally related to the potency of the parent glucocorticoid. Taken together, these data indicate that these types of dual acting cationic lipids can be synthesized with the appropriate starting steroid to tailor activities as desired.

Conceptual and technical aspects of transfection and gene delivery.

Genetically modified animals are state of the art in biomedical research as gene therapy is a promising perspective in the attempt to cure hereditary diseases. Both approaches have in common that modified or corrected genetic information must be transferred into cells in general or into particular cell types of an organism. Here we give an overview of established and emerging methods of transfection and gene delivery and provide conceptual and technical advantages and drawbacks of their particular use. Additionally, based on a flow chart, we compiled a rough guideline to choose a gene transfer method for a particular field of application.

Unexpected transcellular protein crossover occurs during canonical DNA transfection.

Transfection of DNA has been invaluable for biological sciences, yet the effects upon membrane homeostasis are far from negligible. Here, we demonstrate that Neuro2A cells transfected using Lipofectamine LTX with the fluorescently coupled Botulinum serotype A holoenzyme (EGFP-LcA) cDNA express this SNAP25 protease that can, once translated, escape the transfected host cytosol and become endocytosed into untransfected cells, without its innate binding and translocation domains. Fluorescent readouts revealed moderate transfection rates (30-50%) while immunoblotting revealed a surprisingly total enzymatic cleavage of SNAP25; the transgenic protein acted beyond the confines of its host cell. Using intracellular dyes, no important cytotoxic effects were observed from reagent treatment alone, which excluded the possibility of membrane ruptures, though noticeably, intracellular acidic organelles were redistributed towards the plasma membrane. This drastic, yet frequently unobserved, change in protein permeability and endosomal trafficking following reagent treatment highlights important concerns for all studies using transient transfection.

Transfection in Primary Cultured Neuronal Cells.

Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

Establishment of CRISPR-Cas9 ribonucleoprotein mediated MSTN gene edited pregnancy in buffalo: Compare cells transfection and zygotes electroporation.

Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage, transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.

Purification and Transfection Methods of Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of germ cells through which the genome is passed to the next generations. PGCs are found in different locations and variable numbers in the chick embryo, as in other species, depending on the developmental stages. Here, we describe in detail a method based on the Percoll gradient, routinely used in our laboratory, allowing us to obtain from blood and gonad anlages significant numbers of viable PGCs which can be successfully cultured or efficiently genetically modified.

Possibilities and efficiency of MSC co-transfection for gene therapy.

Mesenchymal stem/stromal cells (MSCs) are not only capable of self-renewal, trans-differentiation, homing to damaged tissue sites and immunomodulation by secretion of trophic factors but are also easy to isolate and expand. Because of these characteristics, they are used in numerous clinical trials for cell therapy including immune and neurological disorders, diabetes, bone and cartilage diseases and myocardial infarction. However, not all trials have successful outcomes, due to unfavourable microenvironmental factors and the heterogenous nature of MSCs. Therefore, genetic manipulation of MSCs can increase their prospect. Currently, most studies focus on single transfection with one gene. Even though the introduction of more than one gene increases the complexity, it also increases the effectivity as different mechanism are triggered, leading to a synergistic effect. In this review we focus on the methodology and efficiency of co-transfection, as well as the opportunities and pitfalls of these genetically engineered cells for therapy.