Green Fluorescent Protein Fluorescence Research Articles

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of β-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

Molecular Cloning, Expression and Transport Activity of SaNPF6.3/SaNRT1.1, a Novel Protein of the Low-Affinity Nitrate Transporter Family from the Euhalophyte Suaeda altissima (L.) Pall.

The SaNPF6.3 gene, a putative ortholog of the dual-affinity nitrate (NO3-) transporter gene AtNPF6.3/AtNRT1.1 from Arabidopsis thaliana, was cloned from the euhalophyte Suaeda altissima. The nitrate transporting activity of SaNPF6.3 was studied by heterologous expression of the gene in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the original nitrate transporter. Expression of SaNPF6.3 in Δynt1 cells rescued their ability to grow on the selective medium in the presence of nitrate and absorb nitrate from this medium. Confocal laser microscopy of the yeast cells expressing the fused protein GFP-SaNPF6.3 revealed GFP (green fluorescent protein) fluorescence localized predominantly in the cytoplasm and/or vacuoles. Apparently, in the heterologous expression system used, only a relatively small fraction of the GFP-SaNPF6.3 reached the plasma membrane of yeast cells. In S. altissima plants grown in media with either low (0.5 mM) or high (15 mM) NO3-; concentrations, SaNPF6.3 was expressed at various ontogenetic stages in different organs, with the highest expression levels in roots, pointing to an important role of SaNPF6.3 in nitrate uptake. SaNPF6.3 expression was induced in roots of nitrate-deprived plants in response to raising the nitrate concentration in the medium and was suppressed when the plants were transferred from sufficient nitrate to the lower concentration. When NaCl concentration in the nutrient solution was elevated, the SaNPF6.3 transcript abundance in the roots increased at the low nitrate concentration and decreased at the high one. We also determined nitrate and chloride concentrations in the xylem sap excreted by detached S. altissima roots as a function of their concentrations in the root medium. Based on a linear increase in Cl- concentrations in the xylem exudate as the external Cl- concentration increased and the results of SaNPF6.3 expression experiments, we hypothesize that SaNPF6.3 is involved in chloride transport along with nitrate transport in S. altissima plants.

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.

Tissue-specific in vivo transformation of plasmid DNA in Neotropical tadpoles using electroporation.

Electroporation is an increasingly common technique used for exogenous gene expression in live animals, but protocols are largely limited to traditional laboratory organisms. The goal of this protocol is to test in vivo electroporation techniques in a diverse array of tadpole species. We explore electroporation efficiency in tissue-specific cells of five species from across three families of tropical frogs: poison frogs (Dendrobatidae), cryptic forest/poison frogs (Aromobatidae), and glassfrogs (Centrolenidae). These species are well known for their diverse social behaviors and intriguing physiologies that coordinate chemical defenses, aposematism, and/or tissue transparency. Specifically, we examine the effects of electrical pulse and injection parameters on species- and tissue-specific transfection of plasmid DNA in tadpoles. After electroporation of a plasmid encoding green fluorescent protein (GFP), we found strong GFP fluorescence within brain and muscle cells that increased with the amount of DNA injected and electrical pulse number. We discuss species-related challenges, troubleshooting, and outline ideas for improvement. Extending in vivo electroporation to non-model amphibian species could provide new opportunities for exploring topics in genetics, behavior, and organismal biology.

Comparison of tumor growth assessment using GFP fluorescence and DiI labeling in a zebrafish xenograft model

ABSTRACT DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Abstract 1214: Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus

Abstract Objective: We aimed to establish reversibly immortalized cell lines from human uterine and ovary cells using the Sendai virus (SeV) vector. The immortalized cells derive from normal and benign ovarian epithelial cells and endometrial epithelial cells. Furthermore, we sought to elucidate the mechanisms of carcinogenesis using the immortalized cell lines. Methods: Cells were collected at the time of surgery after obtaining patient consent. The cells used in this study were as follows: ovarian epithelial cells (normal epithelium, Ov n; normal epithelium with germline BRCA1 or BRCA2 mutation, Ov BRCA1 2; ovarian endometrioma, Ov endo; mucinous cystadenoma; Ov m), normal fallopian tube (FT) cells, and endometrial epithelium (normal epithelium, Em n). These cells were infected with temperature-sensitive SeV vectors carrying three immortalization genes, Bmi-1, hTERT, and SV40T.The presence of infection was confirmed through Green Fluorescent Protein (GFP) and Orange Fluorescent Protein (OFP). Immunoreactivity to the anti-human EpCAM antibody (a marker derived from epithelial carcinoma) in each SeV-infected cell was confirmed through flow cytometry. QH and multicolor FISH staining were performed for karyotyping of metaphase chromosomes in each cell line to determine chromosome number and structural abnormalities. Human transcriptome sequencing analysis was performed with NovaSeq 6000 (Illumina) using total RNA from each cell line. Some genes that showed significant expression in each cell line were subjected to real-time PCR (RT-PCR). Results: We established the immortalized cell lines from human uterine and ovarian tissues. SeV-infected cells exhibited GFP and OFP fluorescence, while non-infected cells did not. SeV infection allowed all primary cell lines to grow for 25 or more passages, while non-infected SeV cells lacked the proliferative capacity and showed senescence-like morphology. SeV-infected cells senesced in a temperature-dependent manner. Ov n SeV- infected cells ion causedshowed a small increase in chromosome structural abnormalities. But, Ov BRCA1 and 2 SeV-infected cells showed larger than thatand . Llong-term passaged cells did not show immune response to anti-human EpCAM antibodies in normal cells. Eleven Three genes were predominantly expressed in Ov BRCA1 and Ov BRCA2 cells and were not expressed in Ov n cells. SomeTwo out of ththe three genesem were found to be predominantly expressed in Ov endo cells compared to the expression in the Ovn cell line. Furthermore, RT-PCR results also indicated substantially higher expression of two genes in Ov BRCA1/2 cells and in Ov endo cells compared to the expression in Ov n cells. Conclusion: We succeeded in the reversible immortalization of endometrial and ovarian epithelial cells by using SeV infection. We identified several candidate genes that may be involved in the oncogenic mechanism of ovarian cancer associated with endometriosis or germline BRCA1 and BRCA2. Citation Format: Masayo Okawa, Hiroaki Komatsu, Kohei Hikino, Yuki Iida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Jun Chikumi, Shinya Sato, Genki Hichiwa, Yasuhiro Kazuki, Kanako Kazuki, Fuminori Taniguchi, Mitsuo Oshimura, Tasuku Harada. Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1214.

Gene expression in Verson's glands of the fall armyworm suggests their role in molting and immunity.

Verson's glands are segmental pairs of dermal glands attached to the epidermis in lepidopteran larvae. They produce macromolecules during intermolt period and empty them during each molt. Morphological, histochemical, developmental, and protein analysis studies have been conducted to determine the functions of Verson's glands. However, the exact role of Verson's glands remains unclear. In our previous study, a strain of transgenic fall armyworm, Spdoptera frugiperda expressing green fluorescence protein (GFP) and Systemic RNA interference defective protein 1 (SID1) from Caenorhabditis elegans was established to improve RNA interference (RNAi) efficiency. Unexpectedly, we found that GFP fluorescence was significantly brighter in Verson's glands than in other tissues. Also, RNAi efficiency improved more in Verson's glands than in other tissues. We took advantage of improved RNAi efficiency to explore the function of Verson's glands. RNA-seq analysis revealed that genes highly expressed in Verson's glands code for cuticular proteins, molting fluid proteins, hemolymph proteins, and antimicrobial peptides. Injection of dsRNA targeting essential genes, inhibitor of apoptosis (IAP), Actin, and vacuolar-type ATPase (VATPase) interfered with Verson's glands growth. These results revealed that Verson's glands may contribute to hemolymph, cuticle, molting fluid, and immune response during molting. This study also provide useful tools for future research in identifying the physiological role of Verson's glands in lepidopteran insects.

A simple and accurate method to quantify real-time contraction of vascular smooth muscle cell in vitro

Vascular smooth muscle cells (VSMCs) constitute the medial layer of the blood vessel wall. Their contractile state regulates blood flow in physiological and pathological conditions. Current methods for assessing the contractility of VSMCs are not amenable to the high-throughput screening of pharmaceutical compounds. This study aimed to develop a method to address this shortcoming in the field. Real-time contraction was visualized in living VSMCs using the exogenous expression of green fluorescent protein (GFP). Image-Pro Plus software (IPPS) was used to measure various morphological cell indices. In phenylephrine-treated VSMCs, GFP fluorescence imaging was more accurate than brightfield imaging or phalloidin staining in representing VSMC morphology, as measured using IPPS. Among the multiple indices of VSMC shape, area and mean-diameter were more sensitive than length in reflecting the morphological changes in VSMC. We developed a new index, compound length, by combining the mean-diameter and length to differentiate contracted and uncontracted VSMCs. Based on the compound length, we further generated a contraction index to define a single-VSMC contractile status as single-VSMC contraction-index (SVCI). Finally, compound length and SVCI were validated to effectively assess cell contraction in VSMCs challenged with U46619 and KCl. In conclusion, GFP-based indices of compound length and SVCI can accurately quantify the real-time contraction of VSMCs. In future, the new method will be applied to high-throughput drug screening or basic cardiovascular research.

Development of fluorescence-labeled antibody for immune checkpoint inhibitor using engineered probiotics

Here, we developed a genetically modified lactic acid bacteria (gmLAB) that produces green fluorescent protein (GFP)-conjugating, anti-programmed death-ligand 1 (PD-L1) single-chain variable fragments (scFv) for use as an anti-cancer device that targets immune checkpoint molecules. Since PD-L1 plays a key role as an immune checkpoint molecule in the tumor microenvironment, inhibition and detection of PD-L1 are important in cancer research. The anti-PD-L1 scFv was designed based on atezolizumab, a humanized IgG1 monoclonal antibody, and integrated into a lactococcal GFP gene expression vector. Gene expression from the constructed gmLAB was confirmed by western blotting and GFP fluorescence. The ability of GFP-conjugating anti-PD-L1 scFv against the target antigen, PD-L1 protein, was shown using an enzyme-linked immunosorbent assay. Finally, the ability to recognize PD-L1-expressing tumor-cell lines was confirmed using flow cytometry and fluorescence microscopy. Our results suggest that the gmLAB could be applied to in vivo imaging in cancer as an affordable diagnostic/treatment tool.Graphical

Immunogenicity of a secreted, C-terminally truncated, form of bovine viral diarrhea virus E2 glycoprotein as a potential candidate in subunit vaccine development

Both current live, attenuated, and killed virus vaccines for bovine viral diarrhea virus (BVDV) have their limitations. Here, we report the development of a BVDV subunit vaccine by (i) the expression of a secreted form of a recombinant E2 glycoprotein using BHK21 cells and (ii) determination of the immune responses in mice. The E2 glycoprotein was modified by deletion of the C-terminal transmembrane anchor domain and fusion to a V5 epitope tag. This allowed detection using anti-V5 monoclonal antibodies together with simple purification of the expressed, secreted, form of E2 from the cell media. Furthermore, we genetically fused green fluorescent protein (GFP) linked to E2 via a Thosea asigna virus 2A (T2A) ribosome skipping sequence thereby creating a self-processing polyprotein [GFP-T2A-BVDV-E2trunk-V5], producing discrete [GFP-T2A] and [E2trunk-V5] translation products: GFP fluorescence acts, therefore, as a surrogate marker of E2 expression, BALB/c mice were inoculated with [E2trunk-V5] purified from cell media and both humoral and cellular immune responses were observed. Our antigen expression system provides, therefore, both (i) a simple antigen purification protocol together with (ii) a feasible strategy for further, large-scale, production of vaccines.

Isolation, Purification, and Culture of Embryonic Melanoblasts from Green Fluorescent Protein-expressing Reporter Mice.

In this article, we provide a method to isolate embryonic melanoblasts from reporter mouse strains. The mice from which these cells are isolated are bred into the ROSA26mT/mG reporter background, which results in green fluorescent protein (GFP) expression in the targeted melanoblast population. These cells are isolated and purified by fluorescence-activated cell sorting using GFP fluorescence. We also provide a method to culture the purified melanoblasts for further analysis. This method yields > 99% purity melanoblasts specifically targeted, and can be used for a variety of studies, including gene expression, clonogenic experiments, and biological assays, such as viability, capacity for directional migration, or differentiation into melanin-producing melanocytic cells.

High throughput optimization of medium composition for Escherichia coli protein expression using deep learning and Bayesian optimization

To improve synthetic media for protein expression in Escherichia coli, a strategy using deep neural networks (DNN) and Bayesian optimization was performed in this study. To obtain training data for a deep learning algorithm, E.coli harvesting a plasmid pRSET/emGFP, which introduces the green fluorescence protein (GFP), was cultivated in 81 media designed using a Latin square in deepwell-scale cultivation. The media were composed of 31 components with three levels. The resultant GFP fluorescence intensities were evaluated using a fluorescence spectrometer, and the intensities were in the range 2.69-7.99× 103. A deep neural network model was used to estimate the GFP fluorescence intensities from the culture media compositions, and accuracy was evaluated using cross-validation with 15% test data. Bayesian optimization using the best DNN model was used to calculate 20 representative compositions optimized for GFP expression. According to the validating cultivation, the simulated GFP expression levels included large errors between the estimated and experimental data. The DNN model was retrained using data from the validating cultivation, and secondary estimations were performed. The secondary estimations fit the corresponding experimental data well, and the best GFP fluorescence intensity was 1.4-fold larger than the best of the initial test composition.

In vitro and in vivo gene transfer in the cloudy catshark Scyliorhinus torazame.

Cartilaginous fishes have various unique physiological features such as a cartilaginous skeleton and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, genetic engineering based on gene functions as well as cellular behavior has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene transfer. First, we examined gene transfer into primary cultured cells from cloudy catshark embryos by lipofection, polyethylenimine (PEI) transfection, adenovirus infection, baculovirus infection, and electroporation. Among the methods tested, lipofection, electroporation, and baculovirus infection enabled the successful transfer of exogenous genes into primary cultured cells. We then attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into muscle cells. Furthermore, we succeeded in GFP transfer into adult tissues by electroporation. The in vitro and in vivo gene transfer methods that worked in this study may open ways for genetic manipulation including knockout experiments and cellular lineage analysis in cartilaginous fishes.

Synthesis Processes, Photoluminescence Mechanism, and the Toxicity of Amorphous or Polymeric Carbon Dots.

Fluorescence is the emission of light following photon absorption. This optical phenomenon has many applications in daily life, such as in LED lamps, forensics, and bioimaging. Traditionally, small-molecule fluorophores were most common, but the types of molecules and particles with compelling fluorescence properties have expanded. For example, green fluorescent protein (GFP) was isolated from jellyfish and won the Nobel prize in 2008 due to its significant utility as a fluorescent biomarker. Using the intrinsic fluorescence of GFP, many previously invisible biological processes and substances can now be observed and studied. Other fluorescent materials have also been developed, greatly expanding the potential applications. Semiconductor quantum dots (QDs), which have bright fluorescence and a narrow bandwidth, are a popular choice for display technologies. However, QDs are made of heavy metal elements such as Cd and Se, which pose potential safety concerns to the environment and human health. Thus, new fluorescent organic materials are being developed to mitigate the toxicological concerns while maintaining the QD advantages.One type of new material attracting great attention as an environmentally friendly substitute for semiconductor QDs is carbon dots (CDs). CDs have been developed with strong fluorescence, good photostability, and low toxicity using a variety of precursors, and some synthesis processes have good potential for scale-up. However, since they are made of a variety of materials and through different methods, the structure and properties of CDs can differ from preparation to preparation. There are three major types of CDs: graphene quantum dots (GQDs), carbon quantum dots (CQDs), and amorphous or polymeric carbon dots (PCDs). This Account focuses on PCDs and their unique properties by comparing it with other types of CDs. The synthesis processes, fluorescence properties, fluorescence mechanisms, and toxicity are discussed below with an emphasis on the distinct attributes of PCDs.PCDs can be synthesized from small molecules or polymers. They have an amorphous or cross-linked polymer structure with bright fluorescence. This fluorescence is possibly due to cross-link-enhanced emission or clusteroluminescence that arises from the through-space interactions of heteroatomic-rich functional groups. Other fluorescence mechanisms of CDs, including distinct contributions from the carbon core and surface states, may also contribute. The toxicological profiles of CDs are influenced by the chemical composition, surface functionalization, and light illumination. CDs are generally thought to be of low toxicity, and this can be further improved by removing toxic byproducts, functionalizing the surface, and reducing light exposure to minimize the generation of reactive oxygen species.

Lipopolysaccharide enhances HSV-1 replication and inflammatory factor release in the ARPE-19 cells

PurposeDuring acute retinal necrosis (ARN), retinal pigment epithelial (RPE) cells could be stimulated by both herpes simplex virus (HSV) and lipopolysaccharide (LPS). We aim to investigate the impact of LPS on HSV-1 infection and inflammatory factors in human retinal pigment epithelial cell lines (ARPE-19 cells). MethodsARPE-19 cells were infected by HSV-1F strain and HSVg4 strain, a modified HSV strain with GFP genes cloned in, for 1 h. Different concentrations of LPS were added. Green fluorescence protein (GFP) of HSVg4 and the infected cell protein 4 (ICP4) expression were observed. Cell culture supernatants were collected to detect 34 kinds of related cytokines and chemokines by multiplex immunoassay assay. ResultsUnder LPS treatment, the cytopathic effect displayed as enlarged multinucleated cells, and the GFP fluorescence intensity and ICP4 expression increased in the HSV-1-infected ARPE-19 cells. HSV-1 infection stimulated cytokines IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-9, IL-12P70, IL-15, IL-18, IL-21, IL-27, TNF-α, IFN-γ and chemokines CXCL1, CXCL8, CXCL10, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11 while LPS further enhanced their expression. ConclusionLPS promoted HSV-1 infection and inflammatory factor release in ARPE-19 cells, indicating that ARN could deteriorate when complicated with endotoxemia.

Establishment of Vero cell lines persistently harboring a yellow fever virus 17D subgenomic replicon

Yellow fever virus (YFV), a member of the genus Flavivirus, family Flaviviridae, is the etiological agent for an acute viral hemorrhagic disease, yellow fever. Although effective live attenuated vaccines based on the strain YFV 17D are currently available, no specific antiviral drug is available, and the disease remains a major public health concern. Hence, the discovery and development of antiviral drugs should lead to great benefits in controlling the disease. To provide a screening platform for antiviral agents targeting YFV RNA translation/replication, we have established and characterized two Vero cell lines that persistently harbor a subgenomic replicon derived from YFV 17D-204 (referred to as replicon cells). The replicon carries YFV nucleotides (1 − 176 and 2382−10,862) and a green fluorescent protein (GFP)-Zeocin resistance fusion gene as a selection marker and indicator of persistent replication. Immunofluorescence analysis revealed that both replicon cells and YFV 17D-infected cells showed similar distribution patterns of viral NS4B protein and replication intermediate, double-stranded RNA. Sequencing analysis of persistent replicons from the two replicon cell lines suggested that their nucleotide sequences did not vary greatly following multiple passages. We examined the effect of five agents, the antiviral cytokines interferon-β and -γ, the nucleoside analog ribavirin, the squalene synthase inhibitor zaragozic acid A, and the antibiotic rifapentine, a recently reported entry and replication inhibitor against YFV, on the persistent replication in the two replicon cell lines. These agents were selected because they inhibited both production of YFV 17D and transient replication of a luciferase-expressing replicon in Vero cells, without greatly affecting cell viability. We found that each of the agents decreased GFP fluorescence in the replicon cells, albeit to varying degrees. The agents other than rifapentine also showed a decrease in viral RNA levels in the replicon cells comparable to that seen for GFP fluorescence. These results indicate that persistent replication is susceptible to each of these five agents, although their mechanisms of action may differ. Taken together, these results provide evidence that translation/replication of the replicon in the replicon cells mimics that of the viral genome upon YFV 17D infection, indicating that the replicon cell lines can serve as a useful tool for high-throughput antiviral drug screening.

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains

BackgroundFed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose.ResultsThe mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h−1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h−1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h−1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L.ConclusionThe combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

Multi-Functionalized Heteroduplex Antisense Oligonucleotides for Targeted Intracellular Delivery and Gene Silencing in HeLa Cells.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind, innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition, the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

UV LIGHT INDUCED FLUORESCENCE RECOVERY OF GFP AFTER PHOTOBLEACHING IN MICROSCOPY IMAGING

Fluorescence microscopy has become one of the most important tools for biologists to visualize and study organelles and molecules in a cell. Fluorescent markers are used to visualize specific molecules. One of the most used markers is green fluorescent protein (GFP), which can be expressed along with a protein of interest. However, it is known that the intensity of fluorescence decreases with observation time. To combat this problem, researchers and companies have developed protocols and additives to mitigate photobleaching. In this study, we tested the effects of the three most used culture media on photobleaching and developed a new approach of short-wavelength fluorescence recovery after photobleaching (FRAP). Photobleaching was analyzed by comparing pixel brightness on images taken with a fluorescence microscope. We determined photobleaching of GFP-expressing cells from images taken with a fluorescence microscope by comparing pixel brightness. Statistical analysis was performed to determine the average bleaching for specific culture media. The culture media analyzed had no significant effect on the photobleaching of GFP. However, a brief UV burst (15 sec) restores 50% of the original fluorescence and neither increases ROS nor decreases cell viability. To avoid artifacts in image analysis and interpretation, our study suggests using this simple method of GFP fluorescence recovery with UV light induced FRAP to extend the fluorescence lifetime and imaging of GFP molecules.

An Efficient Agrobacterium-Mediated Genetic Transformation System for Persimmon (Diospyros kaki Thunb.)

Persimmon (Diospyros kaki Thunb.) is an important fruit crop in East Asia and has great commercial potential due to the high demand for fresh edible fruits or processed dried persimmon, as well as its being a source of raw materials for the chemical industry. Genetic engineering has shown great potential in persimmon fruit improvement and compensated for the limitations of conventional breeding. In this study, we developed an efficient transformation system and in vivo regeneration protocol by using the leaf disc culture of ‘Gongcheng Shuishi’ persimmon through the optimization of different basic media and hormone combinations. Based on the secondary induction system on 1/2DKW (DKW medium) + ZT (2.0 mg/L) + TDZ (0.5 mg/L) and MS (1/2 N) + ZT (2.0 mg/L) + IAA (0.1 mg/L), the callus induction rate and adventitious bud induction rate were 96.0% and 94.0%, respectively, and RNA interference of DkANR (ANR, anthocyanidin reductase) was achieved after transformation, thus resulting in a reduction in proanthocyanidin accumulation. The GFP (green fluorescent protein) fluorescence signal was visualized in the transgenic seedlings with GFP gene cotransformation. A total of 17.9% of the positive regenerated transgenic seedlings with empty vector were confirmed by both hyg gene and 35S promoter PCR analysis. Southern blotting assays showed that 20 positive transgenic persimmon seedlings were obtained from 135 regenerated transformants derived from DkANRi. Altogether, a reliable and highly efficient transformation system was achieved in persimmon, which may contribute to gene function identification and genetic improvement in the future.