Green Fluorescent Protein Fluorescence Intensity Research Articles

A Model for Pharmaceutical Proteins Production from Arabidopsis Suspension Culture

Pharmaceutical proteins production in plant suspension culture has been of interest due to the ease of manipulation, low cost and low allergenic reaction in humans. In this work, the constitutive double CaMV 35S promoter and the abiotic stress-responsive promoter of the Rd29a locus were compared to produce Green Fluorescent Protein (GFP) used as a model for secreted protein in Arabidopsis thaliana suspension culture. Induction of Rd29a inducible promoter by plant hormone abscisic acid (ABA) was also studied. A signal sequence (SS) of a 33 kDa rice protein was added to the constructs to promote secretion. The secretion of GFP into the extracellular spaces of the transgenic seedling was detected under confocal microscope. The fl uorescence intensity of GFP in the suspension culture was measured by fl uorometry. A temporal maximum expression of GFP was observed from the CaMV 35S promoter on day 18. This model can be used as an example to produce pharmaceutical proteins in suspension culture in the future.

Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka

Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.

A modified Tet‐ON system minimizing leaky expression for cell‐type specific gene induction in medaka fish

The Tet-ON system is an important molecular tool for temporally and spatially-controlled inducible gene expression. Here, we developed a Tet-ON system to induce transgene expression specifically in the rod photoreceptors of medaka fish. Our modified reverse tetracycline-controlled transcriptional transactivator (rtTAm) with 5 amino acid substitutions dramatically improved the leakiness of the transgene in medaka fish. We generated a transgenic line carrying a self-reporting vector with the rtTAm gene driven by the Xenopus rhodopsin promoter and a tetracycline response element (TRE) followed by the green fluorescent protein (GFP) gene. We demonstrated that GFP fluorescence was restricted to the rod photoreceptors in the presence of doxycycline in larval fish (9days post-fertilization). The GFP fluorescence intensity was enhanced with longer durations of doxycycline treatment up to 72h and in a dose-dependent manner (5-45μg/ml). These findings demonstrate that the Tet-ON system using rtTAm allows for spatiotemporal control of transgene expression, at least in the rod photoreceptors, in medaka fish.

Novel in vivo system to monitor tRNA expression based on the recovery of GFP fluorescence and its application for the determination of plant tRNA expression

We developed a novel assay system to quantitatively detect amber codon suppression by tRNAs expressed in plant cells. The assay was based on recovery of the expression of the green fluorescent protein (GFP) as a reporter, in which a fourth Lys codon (AAG) was changed to a premature amber codon TAG, designated as GFP/amber. Plasmids carrying GFP/amber, suppressor tRNA, and red fluorescent protein (RFF) as an internal control, respectively, were introduced into onion epidermal cells to monitor cell numbers with GFP and RFP fluorescence. First, an amber suppressor tRNASer from tobacco (NtS2) to suppress a TAG codon in GFP mRNA was examined, leading to the recovery of GFP fluorescence. Second, we used two different tRNAs (i.e., AtY3II-am and AtY3II-amiG7), both of which are intron-containing amber suppressor tRNAsTyr, the former impaired precursor-tRNA splicing but the latter did not, as confirmed previously using two different approaches (Szeykowska-Kulinska and Beier, 1991; Akama and Beier, 2003). As expected, coexpression of GFP/amber with AtY3II-am gave no green fluorescence, but significant fluorescence was observed with AtY3II-amiG7. Then, we applied this system for the analysis of 5′-regulatory sequences of the tRNAGln gene family from Arabidopsis. A 5′-flanking sequence of each of the 17 tRNAGln genes was fused to a coding region of an amber suppressor tRNASer gene (NtS2/amber) and its 3′-flanking sequence. Chimeric tRNASer gene, GFP/amber, and RFP were coexpressed, and the GFP or RFP fluorescence intensity was determined in cells using laser-scanning microscopy. In parallel, 17 kinds of original Arabidopsis tRNAGln genes and their chimeric genes with NtS2/amber were all analyzed in cell-free nuclear extract (Yukawa et al., 1997). Comparison of in vitro and in vivo expression of these chimeric tRNA genes displayed generally similar results, accompanied by a wide range of variance in the expression of each gene. Nevertheless, the expression patterns of several genes were clearly the opposite of each other comparing between the two different system, demonstrating the importance of in vivo systems in the study on tRNA expression in plants.

Spaceflight Promoted Myocardial Differentiation of Induced Pluripotent Stem Cells: Results from Tianzhou-1 Space Mission.

During space travel, exposure to microgravity may have profound influence on the physiological function of mammalian cells. In this study, we took opportunity of the Tianzhou-1 (TZ-1) mission to investigate how spaceflight may affect cardiac differentiation of mouse induced pluripotent stem cells (iPSCs). A bioreactor was engineered to perform cell culturing and the time-lapse imaging experiments on-orbit. Transgenic iPSC lines with either Oct4 or α-myosin heavy chain (αMHC) promoter driving green fluorescent protein (GFP) expression were used to study cardiomyocyte (CM) differentiation in real microgravity. The differentiation status was monitored by GFP fluorescence intensity. Interestingly, compared with cells cultured in identical environment at ground gravity, embryoid bodies (EBs) derived from Oct4 reporter iPSC downregulated GFP significantly quicker in space. Meanwhile, EBs derived from αMHC reporter iPSC activated GFP strongly 4 days after launch (P < 0.05) and lasted for 10 days afterward, indicating robust CM formation. This is the first real-time imaging study of iPSC myocardial differentiation in space. Under our experimental condition, real microgravity enhanced the CM differentiation process of iPSCs. Our study provided rare information about iPSC cardiac differentiation in space. In the future, similar automated stem cell experiments may help to realize personalized cardiac tissue biomanufacturing and drug test during space travel.

Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease.

Gene therapy can be designed to efficiently counter pathological features characteristic of neurodegenerative disorders. Here, we took advantage of the glial fibrillary acidic protein (GFAP) promoter to preferentially enhance transgene expression near plaques composed of amyloid-beta peptides (Aβ), a hallmark of Alzheimer's disease (AD), in the TgCRND8 mouse model of amyloidosis.Methods: The delivery of intravenously injected recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) to the cortex and hippocampus of TgCRND8 mice was facilitated using transcranial MRI-guided focused ultrasound in combination with microbubbles (MRIgFUS), which transiently and locally increases the permeability of the blood-brain barrier (BBB). rAAV1/2 expression of the reporter green fluorescent protein (GFP) under a GFAP promoter was compared to GFP expression driven by the constitutive human beta actin (HBA) promoter.Results: MRIgFUS targeting the cortex and hippocampus facilitated the entry of rAAV1/2 and GFP expression under the GFAP promoter was localized to GFAP-positive astrocytes. Adjacent to Aβ plaques where GFAP is upregulated, the volume, surface area, and fluorescence intensity of the transgene GFP were greater in rAAV1/2-GFAP-GFP compared to rAAV1/2-HBA-GFP treated animals. In peripheral organs, GFP expression was particularly strong in the liver, irrespective of the promoter.Conclusion: The GFAP promoter enhanced transgene expression in proximity of Aβ plaques in the brain of TgCRND8 mice, and it also resulted in significant expression in the liver. Future gene therapies for neurological disorders could benefit from using a GFAP promoter to regulate transgene expression in response to disease-induced astrocytic reactivity.

Protein stabilization with retained function of monellin using a split GFP system

Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.

Real-time monitoring of microdistribution of antibody-photon absorber conjugates during photoimmunotherapy in vivo

Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~0.85mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

Background:Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods.Materials and Methods:At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F’ that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry.Results:The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001).Conclusion:According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Rapid and prodium iodide-compatible optical clearing method for brain tissue based on sugar/sugar-alcohol.

The developed optical clearing methods show great potential for imaging of large-volume tissues, but these methods present some nonnegligible limitations such as complexity of implementation and long incubation times. In this study, we tried to screen out rapid optical clearing agents by means of molecular dynamical simulation and experimental demonstration. According to the optical clearing potential of sugar and sugar-alcohol,we further evaluated the improvement in the optical clearing efficacy of mouse brain samples, imaging depth, fluorescence preservation, and linear deformation. The results showed that drops of sorbitol, sucrose, and fructose could quickly make the mouse brain sample transparent within 1 to 2 min, and induce about threefold enhancement in imaging depth. The former two could evidently enhance the fluorescence intensity of green fluorescent protein (GFP) and prodium iodide (PI) nuclear dye. Fructose could significantly increase the fluorescence intensity of PI, but slightly decrease the fluorescence intensity of GFP. Even though the three agents caused some shrinkage in samples, the contraction in horizontal and longitudinal directions are almost the same.

Abstract 323: Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics

Abstract In recent years, cell-based evaluation of drug-induced phenotypic changes has become an important strategy in high throughput drug screening (HTS). However, due to the lack of clinically relevant in vitro models, phenotype-based screening has not resulted in the identification of as many successful compounds as initially assumed. Thus, an application of the long-known multicellular tumor spheroid (MCTS) model for HTS has recently gained attention, as MCTS mimic the complex in vivo situation more closely than standard monolayer cell cultures. However, due to major difficulties with formation of uniform spheroids in a large scale, application of MCTS for HTS has been limited. We focused our efforts on the development of spheroid-based HTS systems in 384-well format for the identification of molecules targeting different tumor microenvironment characteristics. For spheroid formation we used two colorectal cancer cell lines, HCT116 and HT-29, both constitutively expressing green fluorescent protein (GFP). During the spheroid formation process, culture medium was either changed every 4 days or left unchanged. This approach resulted in the generation of two different spheroid models from the each cell line - one representing tumor regions with the sufficient access to nutrients; the other simulating parts with more harsh conditions. Spheroids formed with medium change were more proliferative and less dependent on oxygen. By measuring spheroid GFP fluorescence intensity we could non-invasively monitor drug action over time. First screens revealed a group of drugs particularly effective in quiescent spheroids compared with proliferating spheroids and monolayer cell cultures. All of the compounds inhibited mitochondrial oxidative phosphorylation. The screen confirmed mitochondrial respiration as a promising target for treatment of dormant cells in nutrient-deprived tumor regions and identified molecules with previously unrecognized potential for further clinical advancement. Another screening system was developed to identify drugs that directly influence the immune components of tumor microenvironment. Spheroids are formed by co-culturing cancer cells with human primary monocytes. Over the culture period, monocytes differentiate into tumor-associated macrophages. First pilot experiments showed promising results and more extensive screens are planned. For characterization of drug effects at the transcriptome level, a spheroid-based gene expression profiling method is under development. The method will enable high throughput analysis of transcripts from spheroids treated with chemical libraries. In conclusion, we here present a novel approach to HTS, which by utilizing diverse MCTS models has a potential to identify molecules directly targeting tumor-specific microenvironment components. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Peter Nygren, Maria Hägg Olofsson, Mats Gustafsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. Spheroid-based high throughput screening for identification of molecules targeting different tumor microenvironment characteristics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2015-323

Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors.

Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.

A novel bicistronic sensor vector for detecting caspase-3 activation

Apoptosis is involved in pathological cell death of a wide range of human diseases. One of the most important biochemical markers of apoptosis is activation of caspase-3. Ability to detect caspase-3 activation early in the pathological process is important for determining the timing for interfering with apoptosis initiation and prevention of cell damage. Techniques allowing detection of caspase-3 activity at a single cell level show increased sensitivity, compared to biochemical assays; therefore, we developed a novel bicistronic caspase-3 sensor vector enabling detection of caspase-3 activity in individual cells. We employed green fluorescent protein (GFP) as a reporter for caspase-3 activation in our constructs and assessed the functionality of the generated constructs in transiently transfected Neuro2A and HEK293 cells under basal conditions and following application of okadaic acid (OA) or staurosporine (STS) to induce apoptosis. To ensure responsiveness of the new sensor vector to active caspase-3, we co-transfected the sensor with plasmid(s) overexpressing active caspase-3 and quantified GFP fluorescence using a plate reader. We observed an increase in GFP expression in cells transfected with the new bicistronic caspase-3 sensor in response to both OA and STS. We also showed a significant increase in GFP fluorescence intensity in cells co-expressing the sensor with the plasmid(s) encoding active caspase-3. We generated a novel bicistronic caspase-3 sensor vector which relies on a transcription factor/response element system. The obtained sensor combines high sensitivity of the single cell level detection with the possibility of automated quantification.

197 INFLUENCE OF PLASMID CONSTRUCTION AND CELL TYPE ON THE GENERATION OF GENETICALLY MODIFIED CELL LINES IN CATTLE

Several factors may influence transgenic animal production efficiency, and among them gene construction and the cell type used are of great importance. For a long time, fetal fibroblasts were largely used in generation of transgenic cattle production by nuclear transfer, however adult cells are very useful for cloning once the genotype of the donor nuclei is known, and derivation of such cells is technically simple, efficient, and reproducible. Thus, this study aimed to evaluate the effect of cell type on the percentage of GFP+ cells and fluorescence intensity, using two plasmids constructs encoding for green fluorescent protein (GFP). Transfections were performed in bovine fetal fibroblasts (FF), adult fibroblasts (AF), and cumulus cells (CC) transfected by use of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for the internalization of FUGW or pEGFPN2 plasmid. Forty-eight hours after transfection, the number and the fluorescence intensity (arbitrary units) of GFP+ cells was measured by flow cytometry (FACSAria, FACSDiva Software, BD Biosciences, Franklin Lakes, NJ). Non-transfected cells were used as controls. Means were compared by the Student–Newman–Keuls test (SNK; P &lt; 0.05). The FUGW plasmid promoted a higher rate of transfection and fluorescence intensity than pEGFPN2 in all cell types evaluated. When the FUGW plasmid was used, higher transfection rates were obtained with fetal fibroblasts (FF: 17.8 ± 2.82; AF: 10.66 ± 0.65, CC: 3.9 ± 1.97), while higher fluorescence intensity was observed in adult fibroblasts (FF: 4542 ± 497.09; AF: 9367.5 ± 3490.9, CC: 3496 ± 2638.92). The pEGFPN2 plasmid showed percentage of transfected cells and fluorescence intensity significantly higher than the control only in cumulus cells (pEGFPN2 – FF: 4.9 ± 0.14 and 206.47 ± 755; AF: 760 and 2.4 ± 0.70 ± 330.92; CC: 3.9 ± 1.97, and 1418 ± 36.06, respectively; control – FF: 0.15 ± 0.07 and 249 ± 6 : 36; AF: 0.15 ± 0.07 588 ± 213.54, and CC 0.05 ± 0 214 ± 0.07, respectively). We conclude that the plasmid construction may influence the overall efficiency in transfected cells; however, the transfection percentage and fluorescence intensity is greatly influenced by the cell type. We suggest that transgenesis of a specific cell type may be enhanced by the proper choice of the expression vector.

Abstract 3781: A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning

Abstract In the search for novel anticancer agents, the established principle is to harness differences between normal and tumor tissue. Thus, most of current chemotherapeutic agents target fast-proliferating cancerous cells. However, over the years such treatment strategies have proven less effective than initially expected. One of the main reasons for this is that conditions present in solid tumors, such as hypoxia, low glucose availability and high concentrations of metabolites, promote quiescent, highly resistant cell phenotypes. In our research, we aimed to target and exploit these tumor-specific conditions. To mimic the harsh conditions in a tumor, we used multicellular tumor spheroids (MCTS), which are known to simulate tumor microenvironment in vitro. We developed a novel method to easily form MCTS in 384-well format. There is only one spheroid per well formed and all are comparable in terms of size and shape. For the MCTS formation, we used colon carcinoma cell line, HCT116, with constitutive expression of green fluorescent protein (GFP). Then, we performed spheroid-based high-throughput drug screening using 1600 clinically active compounds. As a surrogate marker for cell viability, we measured mean spheroid GFP fluorescence intensity complemented with a standard resazurin-based assay. Active hits were tested in dose-response experiments in both spheroid and monolayer setup. We identified 12 compounds, which showed preferential activity against the MCTS model. We tested them in spheroid-based clonogenic assay and identified five compounds, which after 72 hrs treatment resulted in no clonogenicity at concentrations equal to monolayer-based IC50-values. Interestingly, all of these compounds have been previously reported to impair mitochondrial function. Three of them have been also reported as uncouplers of oxidative phosphorylation. We tested the influence of all five compounds on oxygen consumption rate. All, except one compound, caused irreversible shutdown of mitochondrial function. For further experiments, we decided to choose nitazoxanide - a clinically used anti-parasitic agent with excellent pharmacokinetics, bioavailability and safety profile. We show that treatment with nitazoxanide, at concentrations well below what is reached clinically, results in down regulation of c-myc, mTOR and Wnt signaling. Preliminary in vivo experiments show encouraging results. We conclude that MCTS-based screening identifies mitochondria as a potential target for cancer treatment and the anthelmintic drug nitazoxanide as a promising candidate. Citation Format: Wojciech Senkowski, Xiaonan Zhang, Maria Hägg Olofsson, Stig Linder, Rolf Larsson, Mårten Fryknäs. A spheroid-based screen identifies mitochondrial targeting as a promising strategy for cancer treatment and drug repositioning. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2014-3781

Translation of the prion protein mRNA is robust in astrocytes but does not amplify during reactive astrocytosis in the mouse brain.

Prion diseases induce neurodegeneration in specific brain areas for undetermined reasons. A thorough understanding of the localization of the disease-causing molecule, the prion protein (PrP), could inform on this issue but previous studies have generated conflicting conclusions. One of the more intriguing disagreements is whether PrP is synthesized by astrocytes. We developed a knock-in reporter mouse line in which the coding sequence of the PrP expressing gene (Prnp), was replaced with that for green fluorescent protein (GFP). Native GFP fluorescence intensity varied between and within brain regions. GFP was present in astrocytes but did not increase during reactive gliosis induced by scrapie prion infection. Therefore, reactive gliosis associated with prion diseases does not cause an acceleration of local PrP production. In addition to aiding in Prnp gene activity studies, this reporter mouse line will likely prove useful for analysis of chimeric animals produced by stem cell and tissue transplantation experiments.

Intravital Imaging Reveals Differential Roles Of Neutrophilic and Stromal Toll-Like Receptor 4 In Hyper-Acute Bone Marrow Response To Systemic Lipopolysaccharide Challenge

IntroductionNeutrophils are the first defenders of host innate immune response against a variety of pathogens. Produced in large quantities from myeloid progenitor cells in the bone marrow, neutrophils are rapidly mobilized and released from the marrow cavity into the general circulation in response to inflammation and invading pathogens. While how neutrophils are released from the bone marrow as a result of interplay among CXCL12 (osteoblastic) and CXCL1/CXCL2 (perivascular) gradients have been studied at a time scale of hours to days post systemic lipopolysaccharide (LPS) challenge, little is known about how the neutrophil and marrow stroma react on a single-cell level in vivo during the hyper-acute (minutes to hours) phase following LPS challenge. MethodsWe performed intravital two-photon microscopy (TPM) on intact cranium of live, anesthetized mice, in which the green fluorescent protein (GFP) is expressed under the Lysozyme M (LysM) promoter expressed predominantly in neutrophils and monocytes, before and immediately after systemic LPS challenge. The behavior of neutrophils and surrounding stroma was analyzed and correlated with systemic neutrophilic responses. Bone marrow chimeras were generated in order to differentiate TLR4-mediated responses in hematopoietic and radio-resistant stromal elements. Neutralizing antibodies to relevant chemokines responsible for neutrophil mobilizations were used to observe changes in intra-marrow neutrophil response following LPS challenge. ResultsWe observed a 2.5-fold increase in the average instantaneous speed, as well as the formation of dynamic GFP+ cellular clusters in 50% of the marrow-resident neutrophils between 30 minutes to 60 minutes following i.v. administration of 100ng of LPS, with the GFP+ neutrophils achieving a peak average speed of 8.4um/min. This was accompanied by release of neutrophils into the systemic circulation as evidenced by a 40% reduction in the overall GFP fluorescence intensity 90 minutes following LPS challenge. Bone marrow chimera experiments revealed that while intra-marrow migration and cellular clustering are dependent on TLR4 signaling in neutrophils, the release of neutrophils into systemic circulation is facilitated by TLR4 signaling on stromal elements. Blocking CXCR2 signaling on neutrophils resulted in the same phenotype as chimeras expressing TLR4-defective stroma, further revealing that marrow stromal elements modulate TLR4-mediated neutrophil mobilization and release via CXCL1/CXCL2 chemokine signaling during this hyper-acute phase of marrow response to LPS. ConclusionsIntravital imaging provides real-time insights to the mechanisms of dynamic regulation of marrow responses to hyper-acute systemic LPS challenge in vivo at a single-cell level within live hosts, and reveals differential contribution of TLR4 signaling on neutrophils and stroma in the bone marrow's response to systemic LPS challenge. Disclosures:No relevant conflicts of interest to declare.

Green Fluorescent Protein as an Indicator of Cryoinjury in Tissues

The fluorescence intensity of Green Fluorescent Protein (GFP) has previously been demonstrated to be an accurate indicator of cellular viability following cryoinsult in individual GFP-transfected cells. In an attempt to ascertain whether GFP fluorescence intensity may also be used as a viability indicator following cryogenic insults in whole tissues, this study examines the transient fluorescence intensity of GFP-transfected mouse hepatic tissue ex vivo following cryoinsult. The observed trends are compared with diffusion-based models. It was observed that the fluorescence intensity of the exposed tissues exhibited slow exponential decay, while the solution in which the tissues were placed inversely gained fluorescence. This slow decay (~3 h) is in contrast to the rapidly diminished fluorescence intensity (seconds) seen in GFP-cell cultures following cryoinsult. These trends suggest that mass diffusion of GFP in the interstitial space, and ultimately into the surrounding medium, is the primary mechanism which determines the fluorescence loss in cryoinjured tissues. These results suggest GFP-transfected tissues may be effectively used as indicators of cryoinjury, and hence viability, following hypothermal insult provided that a sufficiently long incubation is held before observation. It was found that a meaningful observation (15% reduction in fluorescence) could be made three hours subsequent to cryoinjury for the tissues used in this study.

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.

Study on molecular imaging and radionuclide therapy of human nasopharyngeal carcinoma cells transfected with baculovirus-mediated sodium/iodine symporter gene

The non-invasive imaging and radiotherapy by sodium/iodine symporter (NIS) gene transfer have been widely used for many experiments and some clinical studies. Baculovirus is an efficient tool for gene delivery into mammalian cells in vitro and in vivo. However, the applications of NIS and/or baculovirus in nasopharyngeal carcinoma (NPC) cells have not been reported yet. In this study, two recombinant baculoviruses expressing, respectively, NIS and green fluorescent protein (GFP), both under the control of the cytomegalovirus promoter (Bac-NIS and Bac-GFP) were successfully constructed. The infection efficiency and GFP fluorescence intensity of the human NPC cell line CNE-2Z infected by Bac-GFP at different setting of multiplicity of infection (MOI) were determined by flow cytometry. NIS protein expression was detected by indirect immunofluorescence. The 125I uptake and efflux of infected CNE-2Z cells by Bac-NIS were measured by a γ-counter. The cytotoxicity of baculovirus and sodium butyrate and inhibition of iodine uptake by NaClO4 were examined. The radioactivity and GFP fluorescence intensity in co-infected CNE-2Z cells by Bac-NIS and Bac-GFP were measured. Cell colony formation tests were conducted to evaluate the killing effect of Bac-NIS-mediated 131I. Based on the results, the transduction efficiency of Bac-GFP at the MOI of 200 or 400 reached 91.16 and 94.79%, respectively. NIS protein was expressed accurately on transfected CNE-2Z cell membranes and performed its normal function in iodine transport. Baculovirus had hardly any cytotoxic effects on infected cells, while relatively high concentration of sodium butyrate generated cytotoxicity. The correlation coefficient between the GFP fluorescence intensity and radioactivity in co-infected CNE-2Z cells was 0.917. Treatment coupled Bac-NIS with 131I killed the infected tumour cells dramatically in vitro. These results suggest that baculovirus is an effective vector of the gene delivery into CNE-2Z cells and NIS-mediated iodine transport is a potential approach for molecular imaging and radionuclide therapy of NPC.