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Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h -1 - Relative Fluorescence Unit h -1 ), in comparison to batch cultivation (174.0 RFU h -1 ) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L -1 ., (© 2020 American Institute of Chemical Engineers.)

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the filamentous mold Aspergillus fumigatus.

Fluorescence-based techniques enable researchers to monitor physiologic processes, specifically fungal cell viability and death, during cellular encounters with the mammalian immune system with single event resolution. By incorporating two independent fluorescent probes in fungal organisms either prior to, or ensuing experimental infection in mice or in cultured leukocytes, it is possible to distinguish and quantify live and killed fungal cells to interrogate genetic, pharmacologic, and cellular determinants that shape host-fungal cell outcomes. This chapter reviews the techniques and applications of fluorescent fungal reporters of viability, with emphasis on the North American endemic dimorphic fungus, Blastomyces dermatitidis.

Chromoproteins (CPs) are widely-used visual reporters of gene expression. We previously showed that, for coloration in Escherichia coli, CPs had to be overexpressed and that this caused large fitness costs with the most useful (darkly colored) CPs. These fitness costs were problematic because passage of plasmids encoding darkly colored CPs in liquid culture frequently resulted in loss of color due to mutations. Unexpectedly, an early variant of the monomeric red fluorescent protein 1 (mRFP1) gene that was codon-optimized for E. coli (abbreviated mRFP1E) was found here to be an ideal replacement for CP genes. When we subcloned mRFP1E in the same way as our CP genes, it produced a similarly dark color, yet affected E. coli fitness minimally. This finding facilitated testing of several hypotheses on the cause of CP cytotoxicities by gel electrophoresis and size-exclusion chromatography: toxicities correlated with the combination of amounts of expression, oligomerization and inclusion bodies, not isoelectric point. Finally, a semi-rational mutagenesis strategy created several mRFP1 protein variants with different colors without altering the fitness cost. Thus, these mutants and mRFP1E are suitable for comparative fitness costs between different strains of E. coli. We conclude that our new mRFP1E series overcomes prior limitations of CPs., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic β-cells.

Cholecystokinin 1 receptor (CCK1R) is activated by singlet oxygen ( 1 O 2 ) generated in photodynamic action with sulphonated aluminum phthalocyanine (SALPC) or genetically encoded protein photosensitizer (GEPP) KillerRed or mini singlet oxygen generator (miniSOG). A large number of GEPP with varied 1 O 2 quantum yields have appeared recently; therefore, in the present work, the efficacy of different GEPP to photodynamically activate CCK1R was examined, as monitored by Fura-2 calcium imaging. KillerRed, miniSOG, miniSOG2, singlet oxygen protein photosensitizer (SOPP), flavin-binding fluorescent protein from Methylobacterium radiotolerans with point mutation C71G (Mr4511 C71G ), and flavin-binding fluorescent protein from Dinoroseobacter shibae (DsFbFP) were expressed at the plasma membrane (PM) in AR4-2J cells, which express endogenous CCK1R. Light irradiation (KillerRed: white light 85.3 mW‧cm -2 , 4' and all others: LED 450 nm, 85 mW·cm -2 , 1.5') of GEPP PM -expressing AR4-2J was found to all trigger persistent calcium oscillations, a hallmark of permanent photodynamic CCK1R activation; DsFbFP was the least effective, due to poor expression. miniSOG was targeted to PM, mitochondria (MT) or lysosomes (LS) in AR4-2J in parallel experiments; LED light irradiation was found to all induce persistent calcium oscillations. In miniSOG PM -AR4-2J cells, light emitting diode (LED) light irradiation-induced calcium oscillations were readily inhibited by CCK1R antagonist devazepide 2 nM; miniSOG MT -AR4-2J cells were less susceptible, but miniSOG LS -AR4-2J cells were not inhibited. In conclusion, different GEPP PM could all photodynamically activate CCK1R. Intracellular GEPP photodynamic action may prove particularly suited to study intracellular GPCR.

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models. NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Genetically encoded photoelectric silk that can convert photons to electrons (light to electricity) over a wide visible range in a self-power mode is reported. As silk is a versatile host material with electrical conductivity, biocompatibility, and processability, a photoelectric protein is genetically fused with silk by silkworm transgenesis. Specifically, mKate2, which is conventionally known as a far-red fluorescent protein, is used as a photoelectric protein. Characterization of the electrochemical and optical properties of mKate2 silk allows designing a photoelectric measurement system. A series of in situ photocurrent experiments support the sensitive and stable performance of photoelectric conversion. In addition, as a plasmonic nanomaterial with a broad spectral resonance, titanium nitride (TiN) nanoparticles are biologically hybridized into the silk glands, taking full advantage of the silkworms' open circulatory system as well as the absorption band of mKate2 silk. This biological hybridization via direct feeding of TiN nanoparticles further enhances the overall photoelectric conversion ability of mKate2 silk. It is envisioned that the biologically derived photoelectric protein, its ecofriendly scalable production by transgenic silkworms, and the bioassisted plasmonic hybridization can potentially broaden the biomaterial choices for developing next-generation biosensing, retina prosthesis, and neurostimulation applications., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λ max  = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H 2 O 2 system, but not in sodium hypochlorite., Competing Interests: Declaration of competing interest The authors have declared no financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Regulatory T (Treg) cell-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3 YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3 YFP-Cre mice with Insr fl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F 1 -generation mice showed evidence of ectopic recombination and that ~10% of the F 2 -generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell-specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3 YFP-Cre allele, particularly when studying genes that are systemically expressed., (© 2019 John Wiley & Sons Ltd.)

Bioluminescence is a rapid and cost-saving technology that enables monitoring of bacteria in real time in animal infection models. Here, we report a method for labeling GAS with a set of plasmids we have developed and deposited with Addgene. These plasmids can be used to either integrate firefly luciferase (Ffluc) or red-shifted firefly luciferase (FflucRT) into the GAS genome or to introduce the reporters on plasmids which have been stabilized with a toxin-antitoxin system to prevent loss of plasmid in the absence of antibiotic selection.

Bright bioluminescence of ctenophores is conditioned by Ca 2+ -regulated photoproteins. Although they share many properties characteristic of hydromedusan Ca 2+ -regulated photoproteins responsible for light emission of marine animals belonging to phylum Cnidaria, a substantial distinction still exists. The ctenophore photoproteins appeared to be extremely sensitive to light-they lose the ability for bioluminescence on exposure to light over the entire absorption spectrum. Inactivation is irreversible because keeping the inactivated photoprotein in the dark does not recover its activity. The capability to emit light can be restored only by incubation of inactivated photoprotein with coelenterazine in the dark at alkaline pH in the presence of oxygen. Although these photoproteins were discovered many years ago, only the cloning of cDNAs encoding these unique bioluminescent proteins in the early 2000s has provided a new impetus for their studies. To date, cDNAs encoding Ca 2+ -regulated photoproteins from four different species of luminous ctenophores have been cloned. The amino acid sequences of ctenophore photoproteins turned out to completely differ from those of hydromedusan photoproteins (identity less than 29%) though also similar to them having three EF-hand Ca 2+ -binding sites. At the same time, these photoproteins reveal the same two-domain scaffold characteristic of hydromedusan photoproteins. This review is an attempt to systemize and critically evaluate the data scattered through various articles regarding the structural features of recombinant light-sensitive Ca 2+ -regulated photoproteins of ctenophores and their bioluminescent and physicochemical properties as well as to compare them with those of hydromedusan photoproteins. In addition, we also discuss the prospects of their biotechnology applications.

Insulin regulates multiple hepatic metabolic pathways in a seemingly heterogeneous manner. To understand this heterogeneity, we hypothesized that different subpopulations of hepatocytes have different sensitivity to insulin. To test this hypothesis, we developed a fluorescent reporter in which the insulin-responsive fatty acid synthase (FAS) promoter drove expression of a time-dependent fluorescent protein ("timer") and characterized timer expression in flow-sorted cell populations. In Hepa1c1c7 and AML12 hepatocytes, we found that different cell populations express distinct timer fluorescence following insulin treatment, consistent with cellular heterogeneity in the response to insulin. RNA measurements indicated an enrichment of forkhead box O transcription factors in cells with a greater response to insulin. Moreover, we found evidence of increased Akt activation. These data are consistent with a heterogeneous cellular response to insulin and raise the possibility that these different subpopulations underlie the peculiar pathophysiology of hepatic insulin resistance.

Despite the high incidence of neuropathic pain, its mechanism remains unclear. Oxytocin (OXT) is an established endogenous polypeptide produced in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. OXT, which is synthesized by OXT neurons in the SON and the magnocellular part of the PVN (mPVN), is delivered into the posterior pituitary (PP), then released into the systemic blood circulation. Meanwhile, OXT-containing neurosecretory cells in the parvocellular part of the PVN (pPVN) are directly projected to the spinal cord and are associated with sensory modulation. In this study, the OXT system in the hypothalamo-neurohypophysial and hypothalamo-spinal pathway was surveyed using a rat neuropathic pain model induced by partial sciatic nerve ligation (PSL). In the present study, we used transgenic rats expressing an OXT-monomeric red fluorescent protein 1 (mRFP1) fusion gene. In a neuropathic pain model, mechanical allodynia was observed, and glial cell activation was also confirmed via immunohistochemistry. In this neuropathic pain model, a significant increase in the OXT-mRFP1 expression was observed in the PP, the SON, mPVN, and pPVN. Furthermore, OXT-mRFP1 granules with positive fluorescent reaction were remarkably increased in laminae I and II of the ipsilateral dorsal horn. Although the plasma concentrations of OXT did not significantly change, a significant increase of the mRNA levels of OXT and mRFP1 in the SON, mPVN, and pPVN were observed. These results suggest that neuropathic pain induced by PSL upregulates hypothalamic OXT synthesis and transportation to the OXTergic axon terminals in the PP and spinal cord., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable Chinese hamster ovary (CHO) cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase, and a galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH (pHi) of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4-6.5; trans-Golgi: 5.9-6.0; and cytoplasm: 7.1-7.2). The pHi was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through pHi control., (© 2019 Wiley Periodicals, Inc.)

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.

Genetically encoded calcium indicators (GECIs) have become widely used for Ca 2+ imaging in cultured cells as well as in living organisms. Transduction of microglia with viral vectors encoding GECIs provides a convenient means to label microglia for in vivo Ca 2+ imaging. We describe a method using microglia-specific microRNA-9-regulated viral vector, to label microglial cells with a ratiometric GECI (Twitch-2B). This method enables longitudinal recording of both transient and sustained elevations of Ca 2+ in microglia in live animals.

Vaccinia virus recombinants that express fluorescent proteins have a variety of applications such as the identification of infected cells, efficient screening for genetically modified strains, and molecular characterization of virus replication and spread. The detection of fluorescent proteins and viral-fluorescent fusion proteins by fluorescence microscopy is noninvasive and can be used to describe protein localization in live cells and track the intracellular movement of virus particles. This chapter describes a number of approaches for the construction of plasmids and subsequent generation and isolation of fluorescent recombinant viruses.

Calcium signaling plays a significant role in microglial activation. Genetically encoded calcium indicators (GECI) have been widely used for calcium imaging studies in many brain cell types, including neurons, astrocytes, and oligodendrocytes. However, microglial calcium imaging approaches have been hampered by idiosyncrasies of their gene expression and malleable cell properties. The generation of PC::G5-tdT, a Polr2a locus-based conditional mouse reporter of calcium, facilitated the deployment of GECI in microglia. When crossed with the Iba1(Aif1)-IRES-Cre line, all brain microglia of the progeny are labeled with the calcium indicator variant GCaMP5G and the red fluorescent protein tdTomato. This reporter system has enabled in vivo studies of intracellular calcium in large microglial cell populations in cerebral pathologies such as ischemic stroke. In this chapter, we outline specific guidelines for genetic, surgical, imaging, and data analysis aspects of microglial calcium monitoring of the ischemic cortex following middle cerebral artery occlusion.

The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery., (© 2018 Wiley Periodicals, Inc.)

Directed evolution-based protein engineering usually generates large library contained insoluble mutants because of structural disturbance by mutation. To reduce the workload and costs, it is crucial to identify and eliminate those insoluble variants prior to dedicated analysis. Here, we demonstrate a method to visualize soluble protein mutants by using monomeric red fluorescent protein (mRFP) as a fusion tag. A plasmid was devised to express nicotinic acid mononucleotide adenylyltransferase (NadD) fused with a GGGS-linked mRFP tag at the C-terminus. The plasmid was subjected to site saturation mutagenesis within the nadD gene, used to transform Escherichia coli DH10B competent cells, leading to colonies with different red intensities. It was found that the fluorescence intensity of the cell culture correlated positively with the content of NadD-mRFP mutant in the supernatant. Mutation at position 132 led to a library of which most colonies lost the red phenotype, indicating that the position had a key role for proper protein folding. Similarly, mRFP enabled identification of soluble mutants of other enzymes including 1-deoxy-D-xylulose-5-phosphate reductoisomerase and phosphite dehydrogenase. These data suggested that mRFP can serve as a fusion reporter for visualizing soluble protein mutants to facilitate more efficient library screening in directed evolution.

The tumor microenvironment (TME) contains stromal cells in a complex interaction with cancer cells. This relationship has become better understood with the use of fluorescent proteins for in vivo imaging, originally developed by our laboratories. Spectrally-distinct fluorescent proteins can used for color-coded imaging of the complex interaction of the tumor microenvironment in the living state using cancer cells expressing a fluorescent protein of one color and host mice expressing another-color fluorescent protein. Cancer cells engineered in vitro to express a fluorescent protein were orthotopically implanted into transgenic mice expressing a fluorescent protein of a different color. Confocal microscopy was then used for color-coded imaging of the TME. Color-coded imaging of the TME has enabled us to discover that stromal cells are necessary for metastasis. Patient-derived orthotopic xenograft (PDOX) tumors were labeled by first passaging them orthotopically through transgenic nude mice expressing either green, red, or cyan fluorescent protein in order to label the stromal cells of the tumor. The colored stromal cells become stably associated with the PDOX tumors through multiple passages in transgenic colored mice or non-colored mice. The fluorescent protein-expressing stromal cells included cancer-associated fibroblasts and tumor-associated macrophages. The cancer cells in PDOX models can also be labeled with a telomerase-dependent adenovirus containing the gene for green fluorescent protein. Using this model, specific cancer-cell or stromal-cell targeting by potential therapeutics can be visualized. Color-coded imaging enabled the visualization of apparent fusion of cancer and stromal cells. Color-coded imaging is a powerful tool visualizing the interaction of cancer and stromal cells during cancer progression and treatment., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Probiotic Lactobacillus strains are widely used to benefit human and animal health, although the exact mechanisms behind their interactions with the host and the microbiota are largely unknown. Fluorescent tagging of live probiotic cells is an important tool to unravel their modes of action. In this study, the implementation of different heterologously expressed fluorescent proteins for the labelling of the model probiotic strains Lactobacillus rhamnosusGG (gastrointestinal) and Lactobacillus rhamnosusGR-1 (vaginal) was explored. Heterologous expression of mTagBFP2 and mCherry resulted in long-lasting fluorescence of L. rhamnosusGG and GR-1 cells, using the nisin-controlled expression (NICE) system. These novel fluorescent strains were then used to study in vitro aspects of their microbe-microbe and microbe-host interactions. Lactobacillus rhamnosusGG and L. rhamnosusGR-1 expressing mTagBFP2 and mCherry could be visualized in mixed-species biofilms, where they inhibited biofilm formation by Salmonella Typhimurium-gfpmut3 expressing the green fluorescent protein. Likewise, fluorescent L. rhamnosusGG and L. rhamnosusGR-1 were implemented for the visualization of their adhesion patterns to intestinal epithelial cell cultures. The fluorescent L. rhamnosus strains developed in this study can therefore serve as novel tools for the study of probiotic interactions with their environment., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Local delivery of anticancer agents or gene therapeutics to lung tumors can circumvent side effects or accumulation in non-target organs, but accessibility via the alveolar side of the blood-air barrier remains challenging. Polyplexes based on plasmid and linear polyethylenimine (LPEI) transfect healthy lung tissue when applied intravenously (i.v.) in the mouse, but direct delivery into the lungs results in low transfection of lung tissue. Nevertheless, LPEI could offer the potential to transfect lung tumors selectively, if accessible from the alveolar side. This study combined near infrared fluorescent protein 720 (iRFP720) and firefly luciferase as reporter genes for detection of tumor lesions and transfection efficiency of LPEI polyplexes, after intratracheal microspraying in mice bearing 4T1 triple negative breast cancer lung metastases. Simultaneous flow cytometric analysis of iRFP720 and enhanced green fluorescent protein expression in vitro demonstrated the potential to combine these reporter genes within transfection studies. Polyplex biophysics was characterized by single nanoparticle tracking analysis (NTA) to monitor physical integrity after microspraying in vitro. 4T1 cells were transduced with iRFP720-encoding lentivirus and evaluated by flow cytometry for stable iRFP720 expression. Growth of 4T1-iRFP720 cells was monitored in Balb/c mice by tomographic near infrared imaging, tissue and tumor morphology by computed tomography and magnetic resonance imaging. In 4T1-iRFP720 tumor-bearing mice, intratracheal administration of luciferase-encoding plasmid DNA by LPEI polyplexes resulted in successful tumor transfection, as revealed by bioluminescence imaging.

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type -mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2 fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., (© 2016 Wiley Periodicals, Inc.)

The development of cell-based biosensors that give insight into cell and tissue function in vivo is an attractive technology for biomedical research. Here, the development of a cell line expressing a fluorescent calcium sensor for the study of beta-cell function in vivo is reported. The bioresponsive cell model is based on INS-1E pancreatic beta-cells, stably expressing the genetically encoded cameleon-based fluorescent sensor YC3.6 cyto . Following single-cell selection and expansion, functional testing and in vitro encapsulation experiments are used to identify a suitable clone of INS-1E cells expressing the calcium sensor. This clone is transplanted subcutaneous in mouse using a cell macroencapsulation system based on flat sheet porous membranes. Cells in the implanted capsules are able to respond to glucose in vivo by secreting insulin and thereby contributing to the regulation of glycaemia in the mice. Furthermore, fluorescence imaging of explanted devices shows that encapsulated cells maintain high level expression of YC3.6 cyto in vivo. In conclusion, these data show that encapsulated INS-1E cells stably expressing a genetically encoded calcium sensor can be successfully implanted in vivo, and therefore serve as biosensing element or in vivo model to longitudinally monitor the function of pancreatic beta-cells., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy., (© 2016 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 10 4 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.

We have developed a multi-color, imageable, orthotopic mouse model for individual patients with pancreatic cancer. The tumors are labeled by first passaging them orthotopically through transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP). Passage of the tumors in these colored transgenic mice labels the stromal cells of the tumor. The cancer cells in the PDOX are labeled in situ with GFP by telomerase-dependent adenovirus OBP-401. The models are termed imageable patient-derived orthotopic xenografts (iPDOX). The tumors acquired brightly-fluorescent stromal cells from the transgenic host mice, which were stably associated with the tumors through multiple passages. The colored fluorescent protein-expressing stromal cells included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). This model enables powerful color-coded imaging of the interaction of cancer and stromal cells during tumor progression and treatment., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein.

Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.

Fluorescent proteins (FPs) are widely used as genetically encoded markers for noninvasive and quantitative study of biological processes. Development of biomarkers that fluoresce in the near-infrared spectral range allows the study of animals at a deeper level due to high permeability of tissues to light in this wavelength range, compared to the visible light. For widespread use of FPs, such properties as low molecular weight and the monomer become important. In this paper, we developed a FP called the GAF-FP and based on the chromophore- binding domain of bacterial phytochrome from Rhodopseudomonas palustris (RpBphP1). GAF-FP has a molecular mass of ~ 19 kDa, 2 times lower than that of other FP based on BphPs and 1.4 times less than the commonly used GFP-like proteins. Unlike most other near-infrared FP, GAF-FP is a monomer, has high photostability and its structure can withstand the introduction of small peptide inserts. Moreover, GAF-FP can covalently bind two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is found in mammalian tissues. GAF-FP with BV as a chromophore (GAF-FP—BV) has a main absorption band with a maximum at 635 nm and fluorescence maximum at 670 nm, whereby GAF-FP has a high signal to background ratio even if localized at a depth of several mm below the tissue surface. Apart from the near-infrared absorption band, GAF-FP—BV also has also an absorption band in the violet spectral range with a maximum at 378 nm. This property has been used by us to create a chimeric protein consisting of a modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We have shown that the chimeric protein is capable of resonance energy transfer from the substrate, which is oxidized by luciferase, to chromophore of GAF-FP—BV. In the absence of energy acceptor, RLuc8 catalyzes the cleavage of the substrate with light radiation having a peak of 400 nm. At the same time, as a part of GAF-FP—RLuc8 chimeric protein, the energy from the substrate is transferred to the chromophore of FP and then emitted in the near-infrared spectral range corresponding to GAF-FP fluorescence. These results open the way for the creation of new small near-infrared FPs based on various natural BphPs with a prospect of their wider use in cell and molecular biology.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

Background: The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence., Results: Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins., Conclusions: This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator', a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.

Bacteriophytochrome-based infrared fluorescent protein iRFP was tested as an acceptor for F6rster resonance energy transfer (FRET). Far-red GFP-like fluorescent proteins mKate2, eqFP650, and eqFP670 were used as donors; Bacterial expression vectors encoding donor and acceptor proteins fused by a 17-amino acid linker were.constructed. FRET for purified proteins in vitro was, estimated from increase of the donor emission after digestion of the linker. Among the three constructs tested, the most efficient FRET (approximately 30%) was detected for the eqFP650-iRFP pair.

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation., (Copyright © 2015 The Committee on Space Research (COSPAR). Published by Elsevier Ltd. All rights reserved.)