Green Fluorescent Protein Protein Research Articles

Lack of Cannabinoid Type 2 Promoter Activity in Normal or Injured Kidneys Using a Cnr2-GFP Reporter Mouse.

Introduction: Although cannabinoid type 2 (CB2) receptor activity is known to promote diverse biological functions in the kidney, published data regarding CB2 receptor protein levels and cellular distribution within the kidney is inconsistent. The goal of the present study was to investigate the changes of CB2 in the kidney obtained from mice exposed to various forms of kidney injury using a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous cannabinoid receptor 2 (Cnr2) promoter. Materials and Methods: Kidney injury was established in a genetic mouse model expressing green fluorescent protein (GFP) driven by the endogenous Cnr2 promoter. Kidney injury was initiated by either treatment with different chemicals [cisplatin or lipopolysaccharide (LPS)] or by unilateral ureteral obstruction (UUO). Changes in the detection of GFP were used as a proxy for CB2 levels and localization. Histological changes due to the injury stimuli were observed by time-related, morphological changes in kidney cytoarchitecture and blood parameters, such as serum creatinine levels. Cnr2 mRNA levels were detected by reverse transcription coupled to polymerase chain reaction (RT-PCR) while protein changes in the tissue lysates were measured by Western blot analysis. Cellular localization of GFP was detected by fluorescent microscopy. Results: Our data demonstrated that there was no band or a minimally detectable band for GFP using kidney lysates from vehicle- or cisplatin-treated mice. A similar lack of GFP was detected in the UUO kidney versus the contralateral control kidney. This is consistent with the low, albeit detectable levels of Cnr2 mRNA in the kidney samples from control or cisplatin treatment. In frozen kidney sections from vehicle and cisplatin-treated mice, GFP fluorescence was not detectable in tubular epithelia, glomeruli or blood vessels in the cortex. Instead, GFP was detected in rare cells within the interstitial space. A second chemical injury model using LPS found a similar lack of GFP protein levels and an absence of legitimate GFP fluorescence in the main cell types within the kidney. Conclusion: These findings suggest that Cnr2 promoter activity is minimally active in normal or injured kidneys, and that pharmacological manipulation of CB2 receptors may be associated with receptors being expressed in cells recruited to the kidney.

Rational Design of Untranslated Regions to Enhance Gene Expression

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5′ UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5′ and 3′ UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5′ UTR is 25–30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, “Corn”, in the 3′ UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5′ UTRs and 3′ UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3′ UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

The use of apolipoprotein A-I as a transport form of the green fluorescent protein GFP gene in rat hepatocytes

The aim of this study was to investigate the possibility of using apolipoprotein A-I (apo A-I) as a transport form of the green fluorescent protein (GFP) gene into rat hepatocytes.Material and methods. A culture of isolated rat hepatocytes was used as a model. Apo A-I conjugate with fluorescein isothiocyanate (FITC) was obtained by incubation of apo A-I protein with FITC in carbonate buffer pH 9.5 at a ratio of 12.5 μg FITC per 1 mg of protein. Plasmids for pE-GAG transfection with an integrated GFP gene were enriched in the promoter part with cis-elements of the CC(GCC)3-5 type to enhance complex formation with apo A-I. An inverted fluorescence microscope was used for visual analysis of cell fluorescence.Results and discussion. The paper presents evidence of FITC-labeled apo A-I penetration into the cytoplasm and nuclei of rat hepatocytes by receptor-mediated endocytosis. On this basis, it is proposed an attempt to use apo A-I as a means of targeted delivery of plasmid DNA with an integrated GFP gene into the cell. According to the results of fluorescence microscopy, the use of apo A-I as a plasmid DNA transfection agent led to the accumulation of the GFP protein in the cytoplasm of hepatocytes. No fluorescent protein was observed in the absence of apo A-I.Conclusions. The result obtained may indicate the delivery of the GFP gene to the nuclear apparatus of the cell, its expression and GFP protein synthesis.

Engineering a replication-incompetent viral vector for the delivery of therapeutic RNA in crustaceans.

Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, Macrobrachium rosenbergii nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV(ΔRdRp) carrying the GFP RNA cargo resulting in MrNV(ΔRdRp)-GFP. Upon incorporating MrNV(ΔRdRp)-GFP in the diet of the marine Pacific white shrimp (Penaeus vannamei), MrNV(ΔRdRp) particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within 8 days postadministration of the viral vector, which indicated a lack of MrNV(ΔRdRp)-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.

Tuning Electrostatic Interactions To Control Orientation of GFP Protein Adsorption on Silica Surface.

The adsorption of green fluorescent protein (GFP) on silica surfaces has been the subject of growing interest due to its potential applications in various fields, including biotechnology and biomedicine. In this study, we used all-atom molecular dynamics simulations to investigate the charge-driven adsorption of wild type GFP and its supercharged variants on silica surfaces. The results showed that the positively charged variant of GFP adsorbed on the negatively charged silica surface with minimal loss in its secondary structure. Further studies were conducted to understand the role of surface charge distribution on two other positively charged variants of GFP, and the results showed that the orientation of GFP on silica can be easily tuned by careful mutations of the charged amino acid residues on the GFP. This study provides valuable molecular insights into the role of electrostatic-driven adsorption of GFP and highlights the importance of charge interactions in the adsorption process.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes.

In recent years, dendritic cells (DCs)-based vaccines have been developed to combat HIV-1 infection in preclinical and clinical trials. In this study, mice bone marrow cells-derived DCs were pulsed with the recombinant Nef, heat shock protein 27 (Hsp27) and Hsp27-Nef proteins, and also green fluorescent protein (GFP) as a positive control. Then, new platforms of DCs loaded with HIV-1 Nef and Hsp27-Nef proteins (i.e., DC prime/DC boost, DNA prime/DC boost, and DC prime/protein boost) were used to evaluate immune responses in BALB/c mice. Finally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was investigated to secret cytokines in vitro. Our data indicated that the recombinant Nef (∼30kDa), Hsp27 (∼27kDa), GFP (∼27kDa), and Hsp27-Nef (∼53kDa) proteins were greatly generated in E. coli. Moreover, the modified DCs with the recombinant proteins were prepared in large scale. The results of mice immunization showed the highest levels of antibodies, cytokines, and Granzyme B in heterologous DC prime/protein boost regimen using Hsp27-Nef antigen (DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen). The levels of IFN-γ and IL-10 cytokines in splenocytes isolated from mice immunized with DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen were higher than those in other regimens after exposure to SCR virions. These findings demonstrated the importance of Hsp27 as an adjuvant and heterologous DC prime/ protein boost regimen in improvement of immune responses. Indeed, DC Hsp27-Nef prime/ protein Hsp27-Nef boost regimen can be utilized as a promising candidate for HIV-1 vaccine development.

Steric and Electronic Origins of Fluorescence in GFP and GFP-like Proteins.

Fluorescent proteins have become routine tools for biological imaging. However, their nanosecond lifetimes on the excited state present computational hurdles to a full understanding of these photoactive proteins. In this work, we simulate approximately 0.5 nanoseconds of ab initio molecular dynamics to elucidate steric and electronic features responsible for fluorescent protein behavior. Using green fluorescent protein (GFP) and Dronpa2─widely used fluorescent proteins with contrasting functionality─as case studies, we leverage previous findings in the gas phase and solution to explore the deactivation mechanisms available to these proteins. Starting with ground-state analyses, we identify steric (the distribution of empty pockets near the chromophore) and electronic (electric fields exerted on chromophore moieties) factors that offer potential avenues for rational design. Picosecond timescale simulations on the excited state reveal that the chromophore can access twisted structures in Dronpa2, while the chromophore is largely confined to planarity in GFP. We couple ab initio multiple spawning (AIMS) and enhanced sampling simulations to discover and characterize conical intersection seams that facilitate internal conversion, which is a rare event in both systems. Our AIMS simulations correctly capture the relative fluorescence profiles of GFP and Dronpa2 within the first few picoseconds, and we attribute the diminished fluorescence intensity of Dronpa2, relative to GFP, to flexible chromophore intermediates on the excited state. Furthermore, we predict that twisted chromophore intermediates produce red-shifted intensities in the Dronpa2 fluorescence spectrum. If confirmed experimentally, this spectroscopic signature would provide valuable insights when screening and developing novel fluorescent proteins.

Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways

Introduction: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19.Methods: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging.Results: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73–97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control.Conclusion: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Cytokinesis During the First Division of a Mouse Embryo.

Cell division consists of nuclear division (mitosis for somatic cells and meiosis for germ cells) and cytoplasmic division (cytokinesis). Embryonic developments are highly programmed, and thus, each cellular event during early embryo development is stable. For mouse embryos, the first time of mitosis is completed about 22 h after fertilization. However, it remains unclear when the embryo completes its first cytokinesis. Here, we microinjected only one cell in the 2-cell stage mouse embryos with mRNA, which encodes green fluorescence protein (GFP). By monitoring the GFP protein transport dynamics between the two cells, we demonstrated that the first time of cytokinesis in mouse embryos is completed about 15 h after mitosis, namely 37 h after fertilization. In addition, our results indicate that the cytoplasmic protein transport between daughter cells is very effective, which relies on microtubules instead of microfilaments in 2-cell mouse embryos. These results should enrich people’s understanding of the first cell division and cytoskeleton in mouse embryos and then learn more about the mechanisms of early embryo development in mammals.

Structural Water Molecules Confined in Soft and Hard Nanocavities as Bright Color Emitters.

Molecules confined in the nanocavity and nanointerface exhibit rich, unique physicochemical properties, e.g., the chromophore in the β-barrel can of green fluorescent protein (GFP) exhibits tunable bright colors. However, the physical origin of their photoluminescence (PL) emission remains elusive. To mimic the microenvironment of the GFP protein scaffold at the molecule level, two groups of nanocavities were created by molecule self-assembly using organic chromophores and by organic functionalization of mesoporous silica, respectively. We provide strong evidence that structural water molecules confined in these nanocavities are color emitters with a universal formula of {X+·(OH-·H2O)·(H2O) n-1}, in which X is hydrated protons (H3O+) or protonated amino (NH3 +) groups as an anchoring point, and that the efficiency of PL is strongly dependent on the stability of the main emitter centers of the structural hydrated hydroxide complex (OH-·H2O), which is a key intermediate to mediate electron transfer dominated by proton transfer at confined nanospace. Further controlled experiments and combined characterizations by time-resolved steady-state and ultrafast transient optical spectroscopy unveil an unusual multichannel radiative and/or nonradiative mechanism dominated by quantum transient states with a distinctive character of topological excitation. The finding of this work underscores the pivotal role of structurally bound H2O in regulating the PL efficiency of aggregation-induced emission luminogens and GFP.

The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells

To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition.

As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy.Methods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model.Results: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N6-methyladenosine (m6A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition.Conclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m6A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment.

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

VAV1-GFP Fusion Protein Generated By Intron-Based Genome Editing through CRISPR/Cas9 Leads to Spontaneous Activation in TCR Signaling

Recurrent VAV1 mutations and gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) have been identified in peripheral T-cell lymphoma (PTCL) including angioimmunoblastic T-cell lymphoma (AITL) patients. A common theme of these genetic aberrations is the loss of the auto-inhibitory C-terminal SH3 domain of VAV1 resulting in aberrant activation of VAV1 independent of normal activation events. Although mouse models support VAV1 mutation/fusion as having a driver oncogenic role in the pathogenesis of PTCL, investigations on VAV1 activity in human cells were performed mainly on the Jurkat cell line with exogenous expression of VAV1 fusion proteins. This approach has un-physiological expression of VAV1 and the functions of VAV1 fusion/mutation under normal endogenous regulation need to be explored. In this study, we introduced a fusion gene, similar to what has been observed in PTCL, into the endogenous VAV1 locus. The fusion gene was under normal regulatory controls instead of being over-expressed by a viral vector, thus providing a more accurate assessment of its function in vivo. To simulate VAV1 fusion, we knocked in a green fluorescence protein (GFP) sequence followed by a simian virus 40 (SV40) poly(A) signal into intron 25 of VAV1 locus by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. A homologous DNA repair (HDR) template with tandem homologous sequences of VAV1 gene, GFP gene, and a SV40 transcription poly(A) signal was electroporated into Jurkat cells together with the Cas9/sgRNA ribonucleoprotein (RNP) complex. This knock-in disrupted the transcription of exon 26 and exon 27, resulting in an in-frame fusion protein with GFP fused to the C-terminal of SH2 of VAV1 (VAV1SH2-GFP)(Figure A). Because our guide RNA targeted the intron 25 sequence by CRISPR/Cas9 system, any possible indels caused by non-homologous end joining will occur within the intron and will not change the protein sequence of the wild type VAV1. The GFP expressing cells were isolated from the edited cell population by FACS. The fusion of GFP with VAV1 in the sorted cells was confirmed by western blot (Figure B) and these cells displayed a heterozygous VAV1SH2-GFP fusion/wild type (WT) phenotype that mimicked the VAV1 translocations observed in PTCL patients. Jurkat cells with VAV1SH2-GFP showed spontaneous activation of the T-cell receptor (TCR) signaling pathway. Analysis of signaling events downstream of VAV1 demonstrated increased phosphorylation in ITK, LCK, and subsequent ERK in Jurkat cells with VAV1SH2-GFP compared with WT Jurkat cells by western blot (Figure B). We also observed consistently elevated pERK in Jurkat cells with VAV1SH2-GFP by flow cytometry (Figure C). Notably, this elevation in pERK was spontaneous and independent of TCR stimulation with anti-CD3 antibody. VAV1SH2-GFP fusion protein also led to marked activation of downstream NFAT and NF-κB pathways as shown by Luciferase reporter assays (Figure D). Similarly, the enhanced NFAT and NF-κB pathway activation in Jurkat with the fusion protein was independent of TCR stimulation. Interestingly, with antiCD3 stimulation, Jurkat cells with VAV1SH2-GFP showed significantly lower pERK, NFAT and NF-κB activity compared to WT Jurkat cells with anti-CD3 stimulation. In conclusion, in Jurkat cells genetically edited with VAV1SH2-GFP, spontaneous activation of TCR signaling and subsequently increased NFAT and NF-κB activity were observed. Our findings further support that VAV1 C-terminal SH3 domain plays an important regulatory role in blocking VAV1 activity in the absence of proper activation. Removing C-terminal SH3 domain or replacing it with GFP or other protein relieves this inhibition, allowing spontaneous activation independent of TCR stimulation. Our study also indicates the sensitivity of the TCR signaling pathway to the level of activation and hyperactivation is detrimental. To validate the function of VAV1SH2-GFP in normal T cells, we have also successfully edited primary CD4+ T cells with VAV1SH2-GFP and the TCR signaling pathway in edited primary CD4+ T cell is currently being evaluated with and without anti-CD3/CD28 stimulation. Figure Disclosures No relevant conflicts of interest to declare.

The influence of posttranscription silensing protein-suppressor P19 on the transient gfp gene expression level in aztec tobacco plants (Nicotiana rustica L.)

Aim. Genetic constructs creation for studying the influence effect of the viral posttranscriptional silencing protein suppressor p19 on transient reporter green fluorescent protein (GFP) expression and accumulation. Methods. The Golden Gate molecular cloning method was used to create the genetic constructs; the leafy tissues of the Aztec tobacco plants (Nicotiana rustica L.) were infiltrated with a suspension of Agrobacterium tumefaciens L.; the gfp gene expression level was determined by spectrofluorometric and quantitative protein (Bradford method) assays. Results. As a result of the work, the pSPV2324 genetic construct was created, which contained the reporter gene for the green fluorescent protein gfp and the gene for the synthesis of the viral posttranscriptional silencing protein suppressor p19 and its effect on the accumulation of the recombinant GFP protein was determined. A comparative analysis of the gfp gene expression level without and with the suppressor protein synthesis gene in the genetic vector showed that the fluorescence level of GFP protein in Aztec tobacco tissues was 1.3 times higher during spectrofluorimetric analysis using the p19 suppressor gene construct. Conclusions. The positive effect of the viral suppressor silencing P19 gene on the accumulation of recombinant GFP protein in tissues plants of N. rustica L. was shown for the first time. The increase in GFP protein fluorescence when using the p19 suppressor protein construct in spectrofluorimetric analysis coincides with an increase in the total concentration of total water-soluble proteins and the level fluorescence of GFP protein in their native electrophoretic separation.&#x0D; Keywords: cloning, genetic constructs, transient expression, silencing protein suppressor p19, green fluorescent protein (GFP).

MRNA as a Tool for Gene Transfection in 3D Cell Culture for Future Regenerative Therapy.

A combination of three-dimensional (3D) cell culturing and non-viral gene transfection is promising in improving outcomes of cell transplantation therapy. Herein, gene transfection profiles in 3D cell culture were compared between plasmid DNA (pDNA) and messenger RNA (mRNA) introduction, using mesenchymal stem cell (MSC) 3D spheroids. Green fluorescence protein (GFP) mRNA induced GFP protein expression in 77% of the cells in the spheroids, whereas only 34% of the cells became GFP positive following pDNA introduction. In mechanistic analyses, most of the cells in MSC spheroids were non-dividing, and pDNA failed to induce GFP expression in most of the non-dividing cells. In contrast, both dividing and non-dividing cells became GFP-positive after mRNA introduction, which led to a high overall percentage of GFP-positive cells in the spheroids. Consequently, mRNA encoding an osteogenic factor, runt-related transcription factor 2 (Runx2), allowed in vitro osteogenic differentiation of MSCs in spheroids more efficiently compared to Runx2 pDNA. Conclusively, mRNA exhibits high potential in gene transfection in 3D cell culture, in which the cell division rate is lower than that in monolayer culture, and the combination of mRNA introduction and 3D cell culture is a promising approach to improve outcomes of cell transplantation in future regenerative therapy.

Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation

Мета. Дослідити експресію генів gus та gfp в калюсних експлантах амфідиплоїдної пшениці спельти ( Triticum spelta L.) після Agrobacterium -опосередкованої генетичної трансформації. Методи. Для трансформації було обрано сорт пшениці спельти озимої ‘Європа’. Як експланти використовували калюси, отримані зі зрілих зародків. Прекультивацію калюсів здійснювали на живильному середовищі МС (Мурасіге–Скуга), доповненому 2 мг/л 2,4-Д (2,4-дихлорфеноксиоцтова кислота) та 10 мг/л нітратом срібла. Для генетичної трансформації використовували Agrobacterium tumefaciens Conn., штам GV3101, та генетичну конструкцію з репортерними генами gus (ген бета-глюкуронідази (-glucuronidase)) та gfp (ген зеленого флюоресцентного білка (Green Fluorescent Protein, GFP)). Калюси трансформували шляхом інокуляції з агробактеріями та вакуумною інфільтрацією. Далі їх ко-культивували на середовищі МС із 2 мг/л 2,4-Д та 10 мг/л AgNO 3 , але без антибіотиків. Експресію гена gus перевіряли за допомогою гістохімічного, а гена gfp – візуального аналізу (флуоресценція білка GFP в УФ світлі). Рівні експресії генів gfp та gus оцінювали за допомогою програмного забезпечення ImajeJ. Інтеграцію генів gfp та gus в геном спельти перевіряли методом ПЛР . Результати. Генетична трансформація калюсних експлан­тів спельти шляхом їхньої інокуляції в живильному середовищі з агробактеріями та вакуумною інфільтрацією відбувалась з різною частотою. Рівень експресії гена gus за вакуумної інфільтрації становив 4,66 ± 0,74%, за інокуляції – 4,00 ± 0,91%; а гена gfp за вакуумної інфільтрації – 3,66 ± 0,74%, за інокуляції – 4,66 ± 1,39%. Рівень експресії гена gfp був вищим у разі використання інокуляції з агробактеріями, а гена gus – при ва­ку­умній інфільтрації. За допомогою ПЛР-аналізу було під­тверджено інтеграцію генів gfp та gus в геном калюсів спельти. Довжина ПЛР продукту із праймерами до гена gus становила 240 п. н., а до гена gfp – 717 п. н. Висновки. Використання методів вакуумної інфільтрації та інокуляції для генетичної трансформації спельти дали різні результати. Частота генетичної трансформації коливалась від 3,66 до 4,66%. Agrobacterium -опосередкована генетична трансформація амфідиплоїдної пшениці спельти дозволяє дослідити експресію репортерних генів gus та gfp за використання калюсних експлантів, отриманих із зрілих зародків.

Intracellular Delivery of DNA and Protein by a Novel Cell-Permeable Peptide Derived from DOT1L.

Cellular uptake and intracellular release efficiency of biomacromolecules is low because of hurdles in the cell membrane that result in limited access to intra-cellular targets with few functional effects. Cell-penetrating peptides (CPPs) act as cargo delivery vehicles to promote therapeutic molecule translocation. Here, we describe the novel CPP-Dot1l that not only penetrates by itself, but also mediates cargo translocation in cultured cells, as confirmed by fluorescence microscopy and fluorescence spectrophotometry. We conducted cytotoxicity assays and safety evaluations, and determined peptide-membrane interactions to understand the possible pathway for cargo translocation. Additional nucleic acid and covalently conjugated green fluorescence protein (GFP) studies mediated by CPP-Dot1l were conducted to show functional delivery potential. Results indicate that CPP-Dot1l is a novel and effective CPP due to its good penetrating properties in different cell lines and its ability to enter cells in a concentration-dependent manner. Its penetration efficiency can be prompted by DMSO pretreatment. In addition, not only can it mediate plasmid delivery, but CPP-Dot1l can also deliver GFP protein into cytosol. In conclusion, the findings of this study showed CPP-Dot1l is an attractive pharmaceutical and biochemical tool for future drug, regenerative medicine, cell therapy, gene therapy, and gene editing-based therapy development.

Optimization of base editing in Corynebacterium glutamicum

In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C. glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD₆₀₀ of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7-2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.