A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation

Commonly used reporters rely on a single property,such as the fluorescence of GFP and visible color of anthocyanins,therefore these reporters hardly handle the complicated condition in practice.Betaxanthins are a group of plant natural products derived from the amino acid tyrosine.Its visible yellow-orange color and green fluorescence under blue light make it a promising new reporter.Only two enzymatic reactions are required to convert tyrosine into betaxanthins.Here,we synthesized an open reading frame named Bx that contained all the betaxanthins biosynthetic genes and demonstrated its use as a powerful and efficient reporter in tobacco,carrot,and tomato.

A study of reporters as functional entity in news fuzzy language

Fuzziness, as intrinsic property of natural language, appears to be an extremely pervasive phenomenon in language communication with no exception of news reporting. To some extent, the usage of a great number of fuzzy expressions in news reporting reflects the property of reporter as functional entity. On different occasions, reporters, when reporting news, may play such three kinds of roles as the first information source, the second information source or the virtual interpreter. The different roles-playing determines the pragmatic intention of fuzzy language in news reporting.

A rapid reporter assay for recombinant human brain natriuretic peptide(rhBNP)by GloSensor technology

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGIo-G1 was constructed by transfecting pGloSensorTM 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGIo-G1 showed high precision with intraassay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R^2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay （paired t test,p = 0.630）. All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.

A reporter for noninvasively monitoring gene expression and plant transformation

Reporters have been widely used to visualize gene expression,protein localization,and other cellular activities,but the commonly used reporters require special equipment,expensive chemicals,or invasive treatments.Here,we construct a new reporter RUBY that converts tyrosine to vividly red betalain,which is clearly visible to naked eyes without the need of using special equipment or chemical treatments.We show that RUBY can be used to noninvasively monitor gene expression in plants.Furthermore,we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis.The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

Alpha-fetoprotein-targeted reporter gene expression imaging in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in Eastern Asia, and its incidence is increasing globally. Numerous experimental models have been developed to better our understanding of the pathogenic mechanism of HCC and to evaluate novel therapeutic approaches. Molecular imaging is a convenient and up-to-date biomedical tool that enables the visualization, characterization and quantification of biologic processes in a living subject. Molecular imaging based on reporter gene expression, in particular, can elucidate tumor-specific events or processes by acquiring images of a reporter gene’s expression driven by tumor-specific enhancers/promoters. In this review, we discuss the advantages and disadvantages of various experimental HCC mouse models and we present in vivo images of tumor-specific reporter gene expression driven by an alpha-fetoprotein (AFP) enhancer/promoter system in a mouse model of HCC. The current mouse models of HCC development are established by xenograft, carcinogen induction and genetic engineering, representing the spectrum of tumor-inducing factors and tumor locations. The imaging analysis approach of reporter genes driven by AFP enhancer/promoter is presented for these different HCC mouse models. Such molecular imaging can provide longitudinal information about carcinogenesis and tumor progression. We expect that clinical application of AFP-targeted reporter gene expression imaging systems will be useful for the detection of AFP-expressing HCC tumors and screening of increased/decreased AFP levels due to disease or drug treatment.

Red fluorescent protein (DsRed2), an ideal reporter for cotton genetic transformation and molecular breeding

Genes encoding reporter proteins are used as visual marker-assisted tools in genetic transformation as well as plant breeding. In this study, the red fluorescent protein identified in Discosoma sp. coral(DsRed2) was successfully used as a visual marker for cotton genetic engineering. DsRed2 was successfully expressed in two cotton cultivars,JIN668 and YZ1, driven by the Ca MV-35 S promoter via the Agrobacterium-mediated transformation. Our results suggest that DsRed2 expression provides an early-stage selection tool for the transgenic calli via visual observation. Red fluorescence can be detected not only in callus and somatic embryos but also in most tissues and organs of mature plants. The transgenic line Yz-2-DsRed2 was crossed with four different cotton cultivars to assess the transgene heritability and stability in different genetic backgrounds.The heritability of the red color was highly stable when Yz-2-DsRed2 was used as a male parent. The DsRed2 gene expressed 100% in the F_1 hybrids. To investigate the relationship between DsRed2 transcription and DNA methylation, a methylation-specific PCR approach was applied to the Ca MV-35 S promoter region. The results showed a negative association between DNA methylation level in the promoter region and the transgene transcription.Taken together, these findings suggest DsRed2 a visual reporter gene for cotton genetic transformation and molecular breeding programs.

Reporter gene systems for the identification and characterization of cancer stem cells

Cancer stem cells(CSCs)are tumor cells that share functional characteristics with normal and embryonic stem cells.CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo-and radiotherapy,with important roles in tumor progression and the response to therapy.Thus,a current goal of cancer research is to eliminate CSCs,necessitating an adequate phenotypic and functional characterization of CSCs.Strategies have been developed to identify,enrich,and track CSCs,many of which distinguish CSCs by evaluating the expression of surface markers,the initiation of specific signaling pathways,and the activation of master transcription factors that control stemness in normal cells.We review and discuss the use of reporter gene systems for identifying CSCs.Reporters that are under the control of aldehyde dehydrogenase 1A1,CD133,Notch,Nanog homeobox,Sex-determining region Y-box 2,and POU class 5 homeobox can be used to identify CSCs in many tumor types,track cells in real time,and screen for drugs.Thus,reporter gene systems,in combination with in vitro and in vivo functional assays,can assess changes in the CSCs pool.We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics,demonstrating their value in CSCs research.

A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters

We have developed a novel dual enzyme chemistry called rhAmp®SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudioTM 7 Flex, CFX384TM, IntelliQube®, and Biomark HDTM. In comparison to TaqMan®, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.

Tetracysteine as a Reporter for Gene Therapy

Objective To study the feasibility of using tetracysteine （TC） reporter in gene therapy. Methods Effects of TC reporter and conventional reporter genes encoding green fluorescence protein （GFP） and luciferase （Luc） on expression and function of the therapeutic gene MGMT^P140K were compared. Cytotoxicity and drug resistance were studied by Western blot. TC reporter used in therapy was analyzed by flow cytometry （FCM）. Results The TC reporter had no toxicity to cells and neither affected the expression or activity of therapeutic gene as compared to GFP and Luc. TC could be used in blood sample detection. Conclusion TC is a new kind of reporter gene for lentiviral vector in future gene therapy.

Construction and Identification of the Adenoviral Vector with Dual Reporter Gene for Multimodality Molecular Imaging

Summary： In this study, the recombinant adenovirus （Ad） vector containing dual reporter gene [i.e. human transferrin receptor gene （TFRC） and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance （MR） and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction （PCR） and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector （pAdeno） were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colo- rectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor （TfR） and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirns was constructed successfully, and the virus titer was 1.6x10^10 pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly （P〈0.01）. It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.

Duplex micro RNAs assay based on target-triggered universal reporter hybridization

In this paper, we designed and evaluated a duplex detection strategy for micro RNAs（mi RNAs） using universal probe-based target-triggered double hybridization and fluorescent microsphere-based assay system（x MAP array）. In the absence of target mi RNA, reporter DNA cannot hybridize stably with the immobilized capture DNA due to its low melting temperature. Only after adding target mi RNA, can reporter probe hybridize with capture probe to form a stable three-component complex. This targettriggered stable hybridization makes this method possible for highly selective and sensitive detection of multiple mi RNAs. We exemplified a quantitative detection of duplex mi RNAs with a limit of detection of40 p M. The x MAP array platform holds the potential of extending this approach to simultaneous detection of up to 100 mi RNA targets. Considering the simplicity, rapidity and multiplexing, this work promised a potential detection of multiple mi RNA biomarkers for early disease diagnosis and prognosis.

Synthesis and characterization of reporter molecules embedded core-shell nanoparticles as SERS nanotags

Surface-enhanced Raman scattering(SERS)spectroscopy is presented as a sensitive and spe-cific molecular tool for clinical diagnosis and prognosis monitoring of various diseases including cancer.In order for clinical application of SERS technique,an ideal method of bulk synthesis of SERS nanoparticles is necessary to obtain sensitive,stable and highly reproducible Raman signals.In this contribution,we determined the ideal conditions for bulk synthesis of Raman reporter(Ra)molecules embedded silver-gold core-shell nanoparticles(Au@Ra@AgNPs)using hydroquinone as reducing agent of silver nitrate.By using UV-Vis spectroscopy,Raman spectroscopy and transmission electron microscopy(TEM),we found that a 2∶1 ratio of silver nitrate to hydroquinone is ideal for a uniform silver coating with a strong and stable Raman signal.Through stability testing of the optimized Au@Ra@AgNPs over a two-week period,these SERS nanotags were found to be stable with minimal signal change occurred.The sta-bility of antibody linked SERS nanotags is also crucial for cancer and disease diagnosis,thus,we further conjugated the as-prepared SERS nanotags with anti-EpCAM antibody,in which the stability of bioconjugated SERS nanotags was tested over eight days.Both UV-Vis and SERS spectroscopy showed stable absorption and Raman signals on the anti-EpCAM conju-gated SERS nanotags,indicating the great potential of the synthesized SERS nanotags for future applications which require large,reproducible and uniform quantities in order for cancer biomarker diagnosis and monitoring.

Overview of the reporter genes and reporter mouse models

Reporter genes are widely applied in biotechnology and biomedical research owning to their easy observation and lack of toxicity. Taking advantage of the reporter genes in conjunction with imaging technologies, a large number of reporter mouse models have been generated. Reporter mouse models provide systems that enable the studies of live cell imaging, cell lineage tracing, immunological research and cancers etc. in vivo. In this review, we describe the types of different reporter genes and reporter mouse models including, random reporter strains, Cre reporter strains and ROSA26 reporter strains. Collectively, these reporter mouse models have broadened scientific inquires and provided potential strategies for generation of novel reporter animal models with enhanced capabilities.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.

Chinese Four Big Forests of Steles——by our staff reporter Shao De

The chinese upright stone tablets started building early inQindynasty which were called stone-carving in the Initial stage.After the Eastern Han Dynasty, they were gradually changed name as"tablets" or "steles" with the increasing of carved stones. Greatnumbers of stone tablets standing like trees in forest, were later

Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo

Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

A reporter gene system for screening inhibitors of Wnt signaling pathway

Abnormal activation of canonical Wnt signaling has been associated with various types of cancer.Inhibitory reagents targeting the Wnt signaling have great potential to inhibit the growth of relevant tumors.Here we generated a cell-based screening strategy for identification of antagonists of the Wnt/β-catenin signaling pathway.Stable expression wnt3a was generated in HEK293 cell line,which harbors dual-luciferase reporters.The Wnt signaling in the stably transfected cell line was proved to be very sensitive to(-)-epigallocatechin-3-gallate(EGCG)and lithium chloride(LiCl)treatment,respectively.Natural compounds were screened and a couple of novel inhibitory modulators of the Wnt signaling pathway were obtained.

VISUALIZATION OF HEAD AND NECK CANCER MODELS WITH A TRIPLE FUSION REPORTER GENE

The development of experimental animal models for head and neck tumors generally rely on the biol uminescence imaging to achieve the dynamic monitoring of the tumor growth and metastasis due to the complicated anatomical structures.Since the bioluminescence imaging is largely affected by the intracellular luciferase expression level and external D-luciferin concentrations,its imaging accuracy requires further confirmation.Here,a new triple fusion reportelr gene,which consists of a herpes simplex virus type 1 thymidine kinase(TK)gene for radioactive imaging,a far-red fuorescent protein(mLumin)gene for fuorescent imaging,and a firefly luciferase gene for bioluminescence imaging,was introduced for in vrivo observation of the head and neck tumors through multi-modality imaging.Results show that fuorescence and bioluminescence signals from mLumin and luciferase,respectively,were clearly observed in tumor cells,and TK could activate suicide pathway of the cells in the presence of nucleotide analog-ganciclovir(GCV),demonstrating the effecti veness of individual functions of each gene.Moreover,subcutaneous and metastasis animal models for head and neck tumors using the fusion reporter gene-expressing cell lines were established,allowing multi-modality imaging in vio.Together,the established tumor models of head and neck cancer based on the newly developed triple fusion reporter gene are ideal for monitoring tumor growth,assessing the drug therapeutic efficacy and verifying the effec-tiveness of new treatments.

Enhancing sensitivity of SERRS nanoprobes by modifying heptamethine cyanine-based reporter molecules

Surface enhanced resonance Raman scattering(SERRS)is a physical phenomenon that occurs when the energy of incident light is dose to that of electronic excitation of reporter molecules(RMs)attached on substrates.SERRS has showed great promise in healthcare applications such as tumor diagnosis,image guided tumor surgery and real-time evaluation of therapeutic response due to its ultra-sensitivity,manipulating convenience and easy acessibility.As the most widely used organic near-infrared(NIR)fuorophore,heptamethine cyanines possess the electronic ex-citation energy that is close to the plasmon absorption energy of the gold nano scafolds,which results in the extraordinary enhancement of the SERRS signal.However,the effect of hepta-methine cyanine structure and the gold nanoparticle morphology to the SERRS intensity are barely investigated.This work developed a series of SERRS nanoprobes in which two hepta-methine cyanine derivatives(IR783 and IR780)were used as the RM and three gold nanoparticles(nanorod,nanosphere and nanostar)were used as the substrates.Interestingly,even though IR780 and IR783 possess very similar chemical structure,SERRS signal produced by IR780 was determined as 14 times higher than that of IR783 when the RM concentration was6.5 × 10^(-6) M.In contrast,less than 4.0 fold SERRS signal intensity increase was measured by changing the substrate morphologies.Above experimental results indicate that finely tuning the chemical structure of the heptamethine cyanine could be a feasible way to develop robust SERRS probes to visualize tumor or guide tumor resection with high sensitivity and target to background ratio.

Reporting the Reporter： An Evaluation of Health Reporting Among Radio and Television Outfits in Lagos, Nigeria

Radio and television outfits constitute a subset of the mass media belonging specifically to the broadcast system, and vitally provide information useful for development （among others） to its adherents, consumers, and audiences. Health reporting or health news is an aspect of health communication through which the audiences are informed of all matters relating to their total well-being. In this paper, the authors investigated news reporting among broadcast outfits in Lagos-Nigeria as a measure to monitor or watchdog and report the reporter to his clients. The investigation was in two parts： One looked at his strategies in covering and reporting health in the environment; while the other examined news bulletins to subject its health news to a content analysis. The study revealed that health is a neglected concern in broadcast reporting with trigger or vulture approach/practice among broadcast outfits.