Red Fluorescent Protein Fluorescence Research Articles

Improved production of β-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression

BackgroundProviding sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, β-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application.ResultsThe expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of β-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk).ConclusionWe reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of β-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.

Colon-cancer liver metastasis is effectively targeted by recombinant methioninase (rMETase) in an orthotopic mouse model

BackgroundColorectal cancer liver metastasis (CCLM) is the most frequent cause of death of colorectal cancer. Development of novel new effective therapy is needed for CCLM patients to improve outcome. The aim of the present study was to investigate the efficacy of recombinant methioninase (rMETase) on a CCLM orthotopic mouse model of liver metastasis established using the human colon cancer cell line HT29 expressing red fluorescent protein (RFP). Materials and MethodsOrthotopic CCLM nude mouse models were randomized into two groups: control group (n = 6, PBS 200 µl, i.p., daily); rMETase group (n = 6, 100 units/200 µl, i.p., daily). Tumor volume was measured on day 0 and day 15. Body weight was measured twice a week. All mice were sacrificed on day 15. ResultsrMETase significantly inhibited the increase of the liver metastasis as determined by RFP fluorescence area and intensity (p = 0.016 and 0.015, respectively). There was no significant difference of body weight between either group on any day. ConclusionsThe present study suggests that rMETase has future potential therapy for CCLM in the clinic.

Cas9 conjugate complex delivering donor DNA for efficient gene editing by homology-directed repair

Delivery of the CRISPR ribonucleoprotein (RNP) for homology-directed repair (HDR) has been challenging due to the low efficiency. Herein, we developed a Cas9 conjugate complex system which can induce efficient HDR editing with the use of a minimal amount of carrier material. Cas9 from Streptococcus pyogenes was purified and conjugated with low molecular weight polymer (LP). The Cas9-LP conjugates were complexed with single guide RNA (sgRNA) and donor DNA, which showed greatly enhanced internalization into cells compared to native Cas9 complexes, as well as a high extent of co-localization of Cas9 with sgRNA. The cytotoxicity of Cas9-LP complexes was evaluated, demonstrating low cytotoxicity compared to the conventional lipofectamine formulation. Finally, the treatment of Cas9-LP complexes to HEK293T reporter cell line expressing a mutant red fluorescent protein (RFP) results in efficient base correction of the RFP gene (up to 31%), leading to restoration of RFP expression and fluorescence. We anticipate that the current method can be widely used as a platform for efficient HDR editing via ‘minimal carrier-assisted’ delivery without the aid of any external physical stimuli, which can be potentially applied for in vivo and ex vivo editing of cellular targets.

CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli.

Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.

A test strip platform based on a whole-cell microbial biosensor for simultaneous on-site detection of total inorganic mercury pollutants in cosmetics without the need for predigestion

Mercury pollutants such as mercuric chloride (HgCl2), mercurous chloride (Hg2Cl2) and mercuric ammonium chloride (Hg(NH2)Cl) are often found in cosmetics. Previous attempts at the on-site detection of mercury were hindered by the complicated and dangerous pretreatment procedure of converting various forms of mercury to Hg (II) ions. In this study, a test strip platform was developed based on a whole-cell microbial biosensor for the simultaneous detection of soluble and insoluble inorganic mercury pollutants in cosmetics without the need for predigestion. The genetic circuits with constitutively expressed MerR as sensor proteins and inducible red fluorescent protein (RFP) as the reporter were introduced into Escherichia coli to construct the mercury detection biosensor. The RFP fluorescence intensity of this biosensor showed a excellent linear relationship (R2=0.9848) with the Hg (II) concentration ranging from 50nM to 10μM in Luria-Bertani (LB) broth. Further research indicated that this biosensor could respond not only to Hg (II) ions but also to insoluble Hg2Cl2 and Hg2Cl2. The transcriptomic results confirmed the mercury conversion ability of the whole-cell biosensor from a gene expression perspective. This biosensor was embedded on filter paper to form a test strip, which could be used to determine whether the total inorganic mercury pollutants in cosmetics exceeded 1mg/kg. Therefore, this strip provided a low cost, easy-to-use, and instrument-independent method for the detection of mercury pollution in cosmetics, while this study revealed the unique advantages of microbial biosensors in the automatic bioconversion of targets.

Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells.

Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs.

Abstract 5900: Transforming growth factor-beta (TGF-β) inhibitor modulates cancer stroma in a pancreatic cancer orthotopic mouse model

Abstract Background: The tumor microenvironment (TME) contributes to the recalcitrance of this disease. Cancer-associated fibroblasts (CAFs) are thought to be important for the pancreatic cancer TME. Transforming growth factor-beta (TGF-β) has an important role for CAF induction. The present report describes the efficacy of a TGF-β-inhibitor to modulate pancreatic cancer stroma in an orthotopic mouse model. Materials and methods: BxPC-3 human pancreatic adenocarcinoma cells expressing green fluorescent protein (GFP) were implanted subcutaneously in athymic nude mice expressing red fluorescent protein (RFP). Established dual-color tumors, with GFP cancer cells and RFP stroma, were cut into small fragments and then implanted orthotopically onto the tail of the pancreas of RFP-expressing nude mice. Fourteen mice were randomized into control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and treated group (n = 7, SB431542 (TGF-β inhibitor) 0.3 mg, i.p., weekly, for 3 weeks) when tumor diameters reached to 7 mm. Pancreatic cancer stroma was evaluated weekly for 3 weeks by real time intravital imaging with the FV-1000 confocal microscope. Stroma ratio, the percentage of the RFP fluorescence area from the stromal cells relative to the total fluorescence area, was calculated with ImageJ. Tumors were resected on the 21st day from the initial treatment for histological evaluation. Microvessel density using anti-CD31 antibody and expression of TGF-β were examined by immunohistochemical staining. Results: The GFP cancer cells and RFP stromal cells were distinctly imaged. The stroma ratio significantly decreased in the TGF-β-inhibitor treated mice compared to control untreated mice from day 7 to 21. TGF-β expression and microvessel density were reduced in the treated mice. Conclusions: The present study demonstrated selective anti-stromal and anti-angiogenetic activity of TGF-β inhibitor in a dual-color pancreatic orthotopic mouse model. These results indicate potential benefit of TGF-β inhibitor to facilitate drug delivery and improve chemotherapy of pancreatic cancer. Citation Format: Takashi Murakami, Yukihiko Hiroshima, Kentaro Miyake, Tasuku Kiyuna, Ho Kyoung Hwang, Kei Kawaguchi, Jonathan C. DeLong, Thinzer M. Lwin, Kentaro Igarashi, Ryusei Matsuyama, Ryutaro Mori, Takafumi Kumamoto, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Transforming growth factor-beta (TGF-β) inhibitor modulates cancer stroma in a pancreatic cancer orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5900. doi:10.1158/1538-7445.AM2017-5900

Labeling the Stroma of a Patient-Derived Orthotopic Xenograft (PDOX) Mouse Model of Undifferentiated Pleomorphic Soft-Tissue Sarcoma With Red Fluorescent Protein for Rapid Non-Invasive Imaging for Drug Screening.

Our laboratory pioneered patient-derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient-like, in contrast to the ectopic subcutaneous-transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft-tissue sarcoma (UPS-STS) PDOX model acquired bright RFP-expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non-colored nude mice was non-invasively imageable. A PDOX nude mouse model of UPS-STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently passaged to RFP transgenic mice, and after tumor growth were then passaged to non-transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS-STS PDOX models. UPS-STS PDOX tumors, previously grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non-transgenic nude mice maintained the bright fluorescence and were non-invasively imageable. FV1000 confocal imaging revealed diffusely distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non-transgenic mice. These results demonstrate a powerful method to make the PDOX UPS-STS model brightly fluorescent for non-invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP-labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma-targeting drugs, whereby the stromal cells are a visual target. J. Cell. Biochem. 118: 361-365, 2017. © 2016 Wiley Periodicals, Inc.

Establishment of oct4:egfp transgenic and oct4:egfp /β-actin:DsRed double transgenic medaka lines.

As a model to examine cellular multipotency in fish, we established a medaka transgenic (Tg) Tru.oct4:egfp line carrying the green fluorescence protein (GFP) cDNA under control of the Takifugu rubripes oct4 promoter. In this Tg line, GFP could be used to examine both maternal and zygotic oct4 expression during embryogenesis. In addition, while adult Tg fish did not express GFP in any somatic cells, activation of GFP expression was initiated in regenerating fins after amputation. In vitro, some of the cell populations that migrated from fin explants expressed GFP, implying that GFP could be used to monitor oct4 expression in both embryos and in regenerating tissues in the Tru.oct4:egfp Tg line. Next, crossing with β-actin:DsRed Tg line in which all cells emit red fluorescence by expression of red fluorescent protein (RFP) under the β-actin promoter, we prepared a Tru.oct4:egfp /β-actin:DsRed double Tg line. In the double Tg line, early embryonic cells were +GFP/+RFP double positive. In vitro fin cell culture, a small number of +GFP/+RFP double positive cells could be discriminated from other -GFP/+RFP cells. Thus, when transplanted into wild-type medaka, this double Tg line can be used to trace the fate of the transplanted cells using RFP fluorescence after the loss of GFP expression.

Single‐Cell Calcium Imaging of RFP Labeled Interneurons in the Neocortex of Tbr1‐Deficient Neonatal mice

Cortical development involves initial structuring of network features genetically programmed signaling pathways. Later, ion channel activity helps refine neuronal connections. In this study we examine propagating waves of activity in a mouse genetically engineered to lack the transcription factor T‐box, brain 1 (Tbr1), which is involved in the differentiation of cortical excitatory neurons and cortical patterning. Using calcium imaging, we find that wave activity appears at normal frequencies. Additionally we examine the distribution of cortical inhibitory neurons in Tbr1 deficient cortex using red‐fluorescent‐protein (RFP), and find that these neurons are displaced similarly to what is observed in reeler mice. Sensitivity of waves to blockade of GABA signaling is preserved in Tbr1 deficient mice. Finally, we show using dual green and red‐channel imaging of calcium indicator dye and RFP fluorescence, respectively, that inhibitory interneurons participate in spontaneous calcium signaling at normal frequencies in Tbr1 deficient mice. We identify two distinct types of spontaneous calcium signals: one that is consistent with wave activity, and another that is restricted to single cells and not correlated with neighboring cells, and sensitive to blockers of L‐type calcium channels. These results provide evidence that wave activity is a robust property of developing networks, and establish new methods of recording calcium signals from genetically labeled cell populations. We identify distinct calcium signals in immature interneurons that may serve separate purposes in development.

Quantitative analysis of a ubiquitin-dependent substrate using capillary electrophoresis with dual laser-induced fluorescence.

Protein degradation by the ubiquitin-proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS-dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)-fusion of the ubiquitin substrate ribophorin 1 (GFP-RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP-RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris-CHES buffer (pH 9.0) containing 10 mM SDS. GFP-RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP-RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP-RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS-dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.

Construction of a CTGF and RFP–coexpressed Renal Tubular Epithelial Cell and Its Application on Evaluation of CTGF-specific siRNAs on Epithelial-mesenchymal Transition

To construct a connective tissue growth factor (CTGF) and red fluorescent protein (RFP)-coexpressed renal tubular epithelial cell that can be used to quantitatively evaluate the CTGF-induced epithelial-mesenchymal transition (EMT). The CTGF and RFP coding sequences were linked with a "2A" peptide, cloned into an expressing vector, and the HK-C2AR cell clone, which could express CTGF and RFP, were selected from the HK-2 cells after the recombinant plasmid transfection. Then, the CTGF-induced EMT in this cell line was determined, to further determine the association of RFP fluorescence intensity with the CTGF expression or CTGF-induced EMT. CTGF-specific ribonucleic acid (RNA) interference was also used to reconfirm the association. The constructed HK-C2AR cells could stably express RFP and CTGF proportionally, and the CTGF expressed in the cell line could induce EMT of cells, whereas the RFP expressed in the cell could exhibit bright red fluorescence after excitation. After the silence in CTGF, the RFP expression was also decreased, and the CTGF-induced EMT was also inhibited. The CTGF and RFP-coexpressing renal tubular epithelial cell, HK-C2AR, could be used to quantitatively evaluate CTGF-specific small interfering RNAs on EMT by quantitatively detecting the RFP expression. Also, this cell line could be used to quantitatively determine the mechanism of CTGF-induced EMT in renal tubular epithelial cells.

Coupling reporter expression to respiration detects active as well as dormant mycobacteria in vitro and in mouse tissues

BackgroundMycobacterium tuberculosis is known to slow down its transcriptional activity during dormancy. Hence, while using reporter strains, it is important to couple the reporter gene to a promoter that is strong and sensitive both in active and dormant M. tuberculosis. Since respiration is an indispensable process even in dormant bacteria, validation of the promoters of respiratory chain genes – type II NADH dehydrogenase (Pndh) and adenosine triphosphate (ATP) synthase operon (Patps) – of MTB was undertaken for this purpose. MethodsPutative promoter containing sequences were cloned upstream of a red fluorescent protein (RFP) gene. Mycobacterium smegmatis or M. tuberculosis carrying episomal constructs were validated for growth, fitness and fluorescence in different models in vitro and in vivo. ResultsEither promoter can drive stable and strong expression of RFP in actively growing and dormant M. smegmatis in vitro without significantly affecting growth or viability. Fluorescence due to Pndh and Patps was significantly higher than Phsp60. The fitness of M. tuberculosis H37Rv counterparts was unaffected inside J774 macrophages. In immunocompetent mice, despite an initial attenuation in the lungs, both strains reached loads similar to wild type during chronic infection. In the spleen, the fluorescent strain counts were similar to wild type counts throughout. RFP fluorescence in tissue homogenates was more homogenous among mice due to Pndh compared with Patps. ConclusionsCoupling an appropriate reporter to the promoter of ndh-2 gene of M. tuberculosis can make the reporter expression respiration sensitive and thereby reliably detect both active and dormant populations of the reporter strain.

Lung metastasis targeting by a tumor-killing Salmonella typhimurium mutant.

e13631 Background: We have previously developed a tumor-targeting double auxotroph of Salmonella typhimurium termed A1-R (Cancer Research 66, 7647-7652, 2006; Proc. Natl. Acad. Sci. USA 104, 10170-10174, 2007). A1-R requires arginine and leucine, which disable these bacteria from growing in normal tissue but allow the bacteria to have high antitumor virulence in nude mouse models. In the present study, we have observed the efficacy of A1-R bacteria on Lewis lung cancer-bearing C57 immunocompetent mice. Methods: Lung tumors were obtained after injection of 2×106 Lewis lung cancer cells, expressing red fluorescent protein (RFP), into the tail vein of C57 B16 immunocompetent mice. Three days after tumor cell transplantation, the animals were treated with A1-R bacteria via tail-vein injection. Results: Lung tumor growth was inhibited in the treated group, and the mice had much fewer lung metastases tumors compared to the untreated group. A1-R was also injected into the thoracic cavity of the Lewis lung cancer-bearing C57 mice weekly for three weeks. All animals were sacrificed at the end of the experiment. The excised lungs were weighed and imaged by RFP fluorescence. The lung tumors in the treated group were significantly smaller than in the untreated group. Intrathoracic injection of bacteria allowed 108 bacteria to be administered weekly to the host with no obvious toxicity. Various i.v. doses of A1-R were tested, including bolus treatment with a single i.v. injection, medium dose using weekly i.v. administration, and metronomic treatment using a small dose twice a week, i.v. In contrast to intrathoracic administration of A1-R, bolus treatment was toxic to the host. However, the medium i.v. dose and the i.v. metronomic dosing of A1-R were well tolerated. Conclusions: These results suggest that S. typhimurium A1-R is effective against metastatic tumors in immunocompetent mice which is important for future clinical development of bacterial cancer therapy. No significant financial relationships to disclose.

Transthyretin mouse transgenes direct RFP expression or Cre‐mediated recombination throughout the visceral endoderm

Transthyretin (Ttr) is a thyroid hormone transport protein secreted by cells of the visceral yolk sac and fetal liver in developing embryos, and by hepatocytes and the choroid plexus epithelium of the brain in adult mice. Spatiotemporal localization of Ttr mRNA during embryogenesis suggested that Ttr regulatory elements might drive transgene expression throughout the visceral endoderm of early mouse embryos. We use Ttr cis-regulatory elements to generate Ttr::RFP and Ttr::Cre strains of mice, driving red fluorescent protein (RFP) and a nuclear-localized Cre recombinase, respectively. Visualization of RFP fluorescence in Ttr::RFP transgenics confirms reporter localization throughout the visceral endoderm in early embryos and in the visceral yolk sac and fetal liver of later stage embryos. Using both GFP-based and LacZ-based Cre reporter strains, we demonstrate that in Ttr::Cre transgenics, Cre-mediated recombination occurs throughout the visceral endoderm. The Ttr::Cre strain can therefore be used as a tool for genetic modifications within the visceral endoderm lineage.

Identification of a Putative Nogo‐A Enhancer That is Active during Development

Although Nogo‐A is best known for its role as an inhibitor of axon regeneration in the adult central nervous system (CNS), it also appears to have a role in defining neural identity and/or neural tube induction during development. Fibroblast growth factors (Fgfs) participate in the regulation of neural plate‐specific Nogo‐A expression, but the transcriptional activators and associated enhancer elements involved have yet to be defined.In an effort to identify Nogo‐A specific enhancer elements we searched for evolutionarily conserved regions (ECRs) surrounding the Nogo gene. We identified and isolated 5 ECRs within 250 kb of Nogo that are highly conserved across divergent species. Enhancer‐reporter constructs were generated by inserting ECRs upstream of a minimal TK‐HSV promoter coupled to green fluorescent protein (GFP). These constructs were co‐electroporated with red fluorescent protein (RFP) into whole chicken embryos at the early primitive streak stage (HH4). RFP fluorescence confirmed transfection efficiency, while GFP marked enhancer activity.A 125 bp ECR (Peak 1) showed GFP expression within the neural tube, coincident with Nogo‐A expression. Furthermore, this ECR is also activated in a human teratoma cell line (NTERA) that exhibits neural differentiation and Nogo‐A expression.Thus, we have identified a conserved neural‐specific enhancer element that may regulate Nogo‐A expression during development. Further studies are needed to characterize this regulatory module and define the neural‐related transcriptional activators that interact with this region.

Functional imaging in bulk tissue specimens using optical emission tomography: fluorescence preservation during optical clearing

Optical emission computed tomography (optical-ECT) is a technique for imaging the three-dimensional (3D) distribution of fluorescent probes in biological tissue specimens with high contrast and spatial resolution. In optical-ECT, functional information can be imaged by (i) systemic application of functional labels (e.g. fluorophore labelled proteins) and/or (ii) endogenous expression of fluorescent reporter proteins (e.g. red fluorescent protein (RFP), green fluorescent protein (GFP)) in vivo. An essential prerequisite for optical-ECT is optical clearing, a procedure where tissue specimens are made transparent to light by sequential perfusion with fixing, dehydrating and clearing agents. In this study, we investigate clearing protocols involving a selection of common fixing (4% buffered paraformaldehyde (PFA), methanol and ethanol), dehydrating (methanol and ethanol) and clearing agents (methyl salicylate and benzyl-alcohol-benzyl-benzoate (BABB)) in order to determine a 'fluorescence friendly' clearing procedure. Cell culture experiments were employed to optimize the sequence of chemical treatments that best preserve fluorescence. Texas red (TxRed), fluorescein isothiocyanate (FITC), RFP and GFP were tested as fluorophores and fluorescent reporter proteins of interest. Fluorescent and control cells were imaged on a microscope using a DSred2 and FITC filter set. The most promising clearing protocols of cell culture experiments were applied to whole xenograft tumour specimens, to test their effectiveness in large unsectioned samples. Fluorescence of TxRed/FITC fluorophores was not found to be significantly affected by any of the test clearing protocols. RFP and GFP fluorescence, however, was found to be significantly greater when cell fixation was in ethanol. Fixation in either PFA or methanol resulted in diminished fluorescence. After ethanol fixation, the RFP and GFP fluorescence proved remarkably robust to subsequent exposure to either methyl salicylate or BABB. The optimized optical clearing procedure of ethanol fixation followed by methyl salicylate clearing preserved the fluorescence of constitutive RFP in whole xenograft tumour specimens, about 1 cc in dimension, indicating successful extension from cell plating experiments to whole tissue samples. Finally, the feasibility of imaging the 3D distribution of viable tumour cells (as indicated by the RFP emission) is demonstrated by optical-ECT imaging of cleared xenograft tumours using an in-house system.For more information on this article, see medicalphysicsweb.org

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.

Real-time whole-body imaging of an orthotopic metastatic prostate cancer model expressing red fluorescent protein.

We describe here, a whole-body imageable spontaneous metastatic model of human prostate cancer developed by surgical orthotopic implantation (SOI) and visualized by red fluorescent protein (RFP) expression. Human prostate cancer PC-3 cells were transduced with the pLNCX2-DsRed-2-RFP retroviral vector containing the RFP and neomycin-resistance genes. A stable RFP-expressing PC-3 clone was selected in 800 microg/ml G418 in vitro and injected subcutaneously in nude mice. Stable high-level expression of RFP was maintained in the subcutaneously-growing tumors. To utilize RFP expression for metastasis studies, fragments of the subcutaneously-growing tumor, which were comprised of RFP-expressing cells, were implanted by SOI in the prostate of nude mice. Primary tumor growth, progression, and subsequent lymphatic metastases were visualized in live, intact animals in real time by whole-body RFP fluorescence imaging. In total, 100% of the experimental animals developed lymphatic metastasis, the growth of which was monitored in real time by whole-body imaging. The aggressive lymphatic metastasis in this model reflects one of the major metastatic routes of prostate cancer in human patients. Intravital RFP imaging visualized single cancer cells in the lung and bladder. Open RFP imaging at autopsy visualized extensive primary growth and highly disseminated lymph-node metastases. The long-wavelength emission of RFP enabled high sensitivity and resolution of microscopic tumor growth using appropriate imaging techniques. The model should be useful for the real-time evaluation of novel therapeutics for metastatic prostate cancer.

156. Delivery of Plasmid DNA to the Nucleus Using Non-Viral Gene Transfection in Tumor Cell Lines and Primary Tumors

NovaFECTION (Venn Nova), a standard cationic lipid based transfection method, and Nucleofection (Amaxa), a novel electroporation method, were used to transfect three tumor cell lines (human osteosarcoma U2OS, mouse aggressive neuroblastoma AGN2a, and mouse squamous cell carcinoma SCCVII) and primary AGN2a tumors with a plasmid-based red fluorescent protein (RFP) expression vector. RFP expression was detected by flow cytometry, cell division by CFDA SE staining, and subcellular localization of plasmid by quantitative PCR of the plasmid-encoded neo gene. RFP expression was detected in at least 10% of Nucleofected U2OS cells (0.5 mcg of RFP plasmid or 1011 plasmids /106 cells) within 4 to 6 hours. RFP expression peaked at 12 hours post-Nucleofection. In contrast, RFP expression in NovaFECTED U2OS cells was not detected at 6 hours. NovaFECTED RFP was initially detected in at least 10% of the U2OS cells at 12 hours and peaked at 24 hours. To test the ability to transfect primary AGN2a tumors using these methods, tumors were excised from mice, subjected to collagenase D digestion and viable cells were isolated by density gradient centrifugation. The RFP expression pattern in the transfected primary tumors paralleled that seen with the cultured tumor cell lines: RFP expression began at 5 hours and peaked at 48 hours post-Nucleofection, while NovaFECTION required 24 hours to be initially detected and peaked at 4 days. To determine the relationship between cell division and expression of transfected DNA, three cell lines were CFDA SE stained and Nucleofected with 0.25 mcg of RFP plasmid/106 cells. Rates of cell division differed greatly as Nucleofected U2OS cells divided approximately every 2 hours while AGN2a and SCCVII cells divided only once or twice per 24 hours. For Nucleofected U2OS cells, detection of RFP required 6 hours, and AGN2a and SCCVII cells required 24 hours for detectable expression levels of RFP to be seen. To assess the subcellular localization of plasmid transfected either by Nucleofection or NovaFECTION, the neo gene was amplified by real-time PCR from purified nuclei and compared to total cellular levels. Prior to DNA extraction, cells or nuclei were treated with DNase I to eliminate non-transfected plasmid. In U2OS and SCCVII, greater than 1000 copies of plasmid were detected in the nucleus immediately following Nucleofection. By NovaFECTION, plasmid entry into the nucleus required a minimum of 6 hours, and net plasmid amounts were greater. Therefore, early expression of transfected RFP requires immediate nuclear localization, as demonstrated by Nucleofection. However, nuclear localization does not appear to be sufficient for RFP expression in SCCVII cells. Both U2OS and SCCVII had RFP-vector present in the nucleus at time zero, but only the rapidly dividing U2OS cells demonstrated RFP fluorescence at 6 hours post-Nucleofection. The lack of RFP expression prior to cell division may reflect an as yet undescribed contribution by cell division to plasmid-based gene vector expression.