Erythroid cells, the predominant circulating blood cells, are essential for oxygen and carbon dioxide transport (Obeagu, 2024).Their production, erythropoiesis, involves the coordinated synthesis of globin chains and ...Erythroid cells, the predominant circulating blood cells, are essential for oxygen and carbon dioxide transport (Obeagu, 2024).Their production, erythropoiesis, involves the coordinated synthesis of globin chains and heme molecules to assemble hemoglobin(Zhang et al., 2021). The erythroid-specific enzyme δ-aminolevulinate synthase 2 (ALAS2) is a key rate-limiting factor in heme biosynthesis,with its expression increasing in late-stage erythropoiesis to meet heme demands (Sadlon et al., 1999). Zebrafish (Danio rerio) is a well-established model for studying erythropoiesis due to its genetic tractability and optical transparency (Zhang and Hamza, 2019;Zhang et al., 2021). The Tol2-mediated Gal4-UAS system has been widely applied for gene and enhancer trapping in zebrafish (Asakawa and Kawakami, 2009). However, reliable Gal4 enhancer-trap lines for erythropoiesis remain limited. Here, we report a transgenic zebrafish line with erythroid-specific Gal4FF expression under the control of the endogenous alas2 promoter, offering a more precise erythroblast labeling than the gata1a reporter line. This model provides a valuable tool for erythroid-specific investigations of blood flow dynamics and gene function.展开更多
Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential i...Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.展开更多
The advancement of tissue clearing technology has significantly propelled neuroscience research.Nevertheless,the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for ti...The advancement of tissue clearing technology has significantly propelled neuroscience research.Nevertheless,the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for tissue clearing procedures,resulting in a substantial decrease in fluorescent intensity after clearing.In this study,we developed the Ci1 reporter mouse strain(where Ci stands for the Chinese Institute for Brain Research,CIBR)based on the bright red fluorescent protein mScarlet.The Ci1 reporter exhibits no fluorescence leakage in various organs or tissue types and can be readily crossed with multiple tissue-specific Cre lines.Compared to the Ai14 mouse strain,the Ci1 reporter strain demonstrates lower non-specific leakage,stronger fluorescence intensity in different tissues,and better preservation of fluorescence following tissue clearing treatment.The creation of the Ci1 reporter provides a more effective tool for both neuroscience and other biomedical research applications.展开更多
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...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.展开更多
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...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.展开更多
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 293...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.展开更多
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 pathogeni...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.展开更多
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 s...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.展开更多
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 u...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.展开更多
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-a...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.展开更多
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 （Lu...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.展开更多
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 e...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.展开更多
A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg^2+ as the reporter module.The developed biosens...A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg^2+ as the reporter module.The developed biosensor showed response for Hg^2+ in a relatively wide range of 1–10,000 nmol/L,and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs.In addition,the biosensor could distinguish Hg^2+ easily from multiple metal ions and displayed strong adaptability to extensive p H conditions (pH 4.0–10.0).More importantly,the developed biosensor was able to provide an initial assessment of Hg^2+ spiked in the environmental water with the recoveries between 85.70%and 112.50%.The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other wholecell biosensors,meeting the practical requirements with sufficient sensing performance.展开更多
Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bi...Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bioassay method is needed for determination of dioxins in food samples.In this study,we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples.The samples were extracted by shaking with water/isopropanol(1:1 v/v)and hexane and cleaned-up by a multi layered silica gel column and an alumina column,then analyzed using CBG 2.8 D cell line.We compared the results obtained from the CBG 2.8 D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry(HRGC-HRMS)analysis.Good correlations were observed between these two methods(r^2=0.93).While the slope of regression line was 1.76,the bioanalytical equivalent(BEQ)values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568(the reciprocal of 1.76).In conclusion,CBG 2.8 D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.展开更多
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 ar...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.展开更多
Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and...Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and there are still no available specific anti-YFV medicines.Herein,by introducing the Renilla luciferase gene(Rluc) into an infectious clone of YFV vaccine strain 17 D,we generated a recombinant virus 17 D-Rluc.2 A via reverse genetics approaches.The 17 D-Rluc.2 A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain.Importantly,the reporter luciferase was efficiently expressed in 17 D-Rluc.2 A-infected mammalian and mosquito cells,and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction.Furthermore,by a combination of the 17 D-Rluc.2 A reporter virus and selective 2’-hydroxyl acylation analyzed by primer extension(SHAPE)technology,the conserved 5’-SLA element was shown to be essential for YFV replication,highlighting the capability of17 D-R1 uc.2 A in the investigation of YFV replication.At last,we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17 D-Rluc.2 A-infected cells,demonstrating its potential application in the evaluation of anti-viral medicines.Taken together,the 17 D-Rluc.2 A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.展开更多
Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeut...Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine(HHT), bruceine D(BD), dihydroartemisinin(DHA) and digitonin(DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549.The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.展开更多
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 a...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.展开更多
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 larg...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.展开更多
In this study,a natural cotton thread immunoassay device combined with gold nanorod（GNR） reporter probe is developed for the rapid,sensitive and quantitative electrochemical determination of human ferritin,a lung ca...In this study,a natural cotton thread immunoassay device combined with gold nanorod（GNR） reporter probe is developed for the rapid,sensitive and quantitative electrochemical determination of human ferritin,a lung cancer related biomarker.Human ferritin as an analyte and a pair of monoclonal antibodies are used to demonstrate the proof-of-concept on the cotton thread immunoassay device.An enhancement of the sensitivity is achieved by using gold nanorod as an electroactive report probe compared with a traditional gold nanoparticle（GNP） report probe.The device was capable of measuring 1.58 ng/mL ferritin in 30 min by anodic stripping voltammetry（ASV） testing,which meet the requirement for clinical diagnosis.展开更多
基金supported by the National Key Research and Development Plan of China (2023YFA1802000)the National Natural Science Foundation of China for Distinguished Young Scholars (31925014),the National Natural Science Foundation of China Key Program (32130033),the National Natural Science Foundation of Original Exploratory Program (32350006)+5 种基金the Key Research Program of Frontier Sciences,Chinese Academy of Sciences (ZDBS-LY-SM010)Shanghai Pilot Program for Basic Research-Chinese Academy of Sciences,Shanghai Branch (JCYJ-SHFY-2022-006)Shanghai Science Technology Innovation Action Plan for Basic Research Program (21JC1406300)Haihe laboratory of Cell Ecosystem Innovation Fund (24HHXBSS00011)CAS project for Young Scientists in Basic Research (YSBR-077)supported by Science and Technology Commission of Shanghai Municipality (Shanghai Rising-Star Program,23QA1411300)
文摘Erythroid cells, the predominant circulating blood cells, are essential for oxygen and carbon dioxide transport (Obeagu, 2024).Their production, erythropoiesis, involves the coordinated synthesis of globin chains and heme molecules to assemble hemoglobin(Zhang et al., 2021). The erythroid-specific enzyme δ-aminolevulinate synthase 2 (ALAS2) is a key rate-limiting factor in heme biosynthesis,with its expression increasing in late-stage erythropoiesis to meet heme demands (Sadlon et al., 1999). Zebrafish (Danio rerio) is a well-established model for studying erythropoiesis due to its genetic tractability and optical transparency (Zhang and Hamza, 2019;Zhang et al., 2021). The Tol2-mediated Gal4-UAS system has been widely applied for gene and enhancer trapping in zebrafish (Asakawa and Kawakami, 2009). However, reliable Gal4 enhancer-trap lines for erythropoiesis remain limited. Here, we report a transgenic zebrafish line with erythroid-specific Gal4FF expression under the control of the endogenous alas2 promoter, offering a more precise erythroblast labeling than the gata1a reporter line. This model provides a valuable tool for erythroid-specific investigations of blood flow dynamics and gene function.
基金supported by the first batch of grants from the State Key Laboratory of Drug Regulatory Science,China(Grant No.:2023SKLDRS0108).
文摘Recombinant human growth hormone(rhGH)has been widely used for the treatment of disorders associated with GH deficiency and multiple clinical indications[1].Accurate determination of biological activity is essential in the development,registration,and quality control of rhGH pharmaceutical products[2].However,the existing in vivo bioassay procedure based on somatropin-induced weight gain in rats is complicated,and the use of a rat cell line-based approach(Nb2-11 bioassay),which measures the production of adenosine triphosphate(ATP)as a direct indicator of cell growth,has a low mechanism of action(MOA)relevance.Therefore,novel rhGH bioassays are still needed.To this end,we developed a reporter gene assay(RGA)based on the GH/insulin-like growth factor-1(IGF-1)axis.
基金supported by the startup funding from the Chinese Institute for Brain Research to Hu Zhao.
文摘The advancement of tissue clearing technology has significantly propelled neuroscience research.Nevertheless,the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for tissue clearing procedures,resulting in a substantial decrease in fluorescent intensity after clearing.In this study,we developed the Ci1 reporter mouse strain(where Ci stands for the Chinese Institute for Brain Research,CIBR)based on the bright red fluorescent protein mScarlet.The Ci1 reporter exhibits no fluorescence leakage in various organs or tissue types and can be readily crossed with multiple tissue-specific Cre lines.Compared to the Ai14 mouse strain,the Ci1 reporter strain demonstrates lower non-specific leakage,stronger fluorescence intensity in different tissues,and better preservation of fluorescence following tissue clearing treatment.The creation of the Ci1 reporter provides a more effective tool for both neuroscience and other biomedical research applications.
文摘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.
基金supported by grants from the National Transgenic Science and Technology Program(2019ZX08010-003,2019ZX08010-001)to Y.H.and H.Z.T.Z.is a TIGS postdoctoral fellow.W。
文摘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.
基金supported by grants from the National Science and Technology Major Project(No.2015ZX09501008-001)the Middle-aged and Young Development Research Foundation of NIFDC(No.2017B3)
文摘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.
基金Supported by Korea Science and Engineering Foundation,No.2012M2A2A7013480 and No.2013M2C2A1074238
文摘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.
基金supported by National Key Research and Development Program(2016YFD0100203-9)National R&D Project of Transgenic Crops of Ministry of Science and Technology of China(2016ZX08010001-006)+1 种基金Program of Introducing Talents of Discipline to Universities in China(B14032)Fundamental Research Funds for the Central Universities(2013PY064,2662015PY028,2662015PY091)
文摘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.
文摘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.
基金Supported by UNAM-PAPIIT,No.IN219719 and No.IA205421CONACYT,No.A1-S-18285.
文摘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.
文摘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.
基金supported by grants from the National Basic Research Program(973 program)(No.2011CB935800)the National Natural Science Foundation of China(Nos.81130027,81071204)
文摘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.
基金supported by the National Natural Science Foundation of China (No.31800631)the Natural Science Foundation of Guangxi Province (No.2018JJB120049)+1 种基金the Middleaged and Young Teachers’ Basic Ability Promotion Project of Guangxi (No.2018KY0361)the BAGUI Scholar Program of Guangxi Province of China。
文摘A signal-amplified mercury sensing biosensor with desired sensitivity was developed through firstly using the GFP mutant with fluorescence increasing response towards Hg^2+ as the reporter module.The developed biosensor showed response for Hg^2+ in a relatively wide range of 1–10,000 nmol/L,and the detection limit was improved one or two orders of magnitude in comparison with most metal-sensing biosensors in similar constructs.In addition,the biosensor could distinguish Hg^2+ easily from multiple metal ions and displayed strong adaptability to extensive p H conditions (pH 4.0–10.0).More importantly,the developed biosensor was able to provide an initial assessment of Hg^2+ spiked in the environmental water with the recoveries between 85.70%and 112.50%.The signal-amplified strategy performed by the modified reporter module will be widely applicable to many other wholecell biosensors,meeting the practical requirements with sufficient sensing performance.
基金the National Natural Science Foundation of China(Nos.21525730 and 21527901)the National Key Research and Development Program of China(No.2018YFA0901103)。
文摘Food,especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans(PCDD/Fs),and dioxin-like polychlorinated biphenyls(dl-PCBs)for human exposure.So,a simple,rapid and cheap bioassay method is needed for determination of dioxins in food samples.In this study,we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples.The samples were extracted by shaking with water/isopropanol(1:1 v/v)and hexane and cleaned-up by a multi layered silica gel column and an alumina column,then analyzed using CBG 2.8 D cell line.We compared the results obtained from the CBG 2.8 D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry(HRGC-HRMS)analysis.Good correlations were observed between these two methods(r^2=0.93).While the slope of regression line was 1.76,the bioanalytical equivalent(BEQ)values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568(the reciprocal of 1.76).In conclusion,CBG 2.8 D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.
基金financially supported by the National Science Foundation of China (Grant No. 21575029)
文摘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.
基金This work is supported by the National Natural Science Foundation of China(81871632 and 32070183)the Natural Science Foundation of Guangdong Province(2020A1515010656)+1 种基金the Creative Research Group Foster Project of the Sun Yat-sen Universitysupported by the One-Hundred People Project of the Sun Yat-sen University。
文摘Yellow fever virus(YFV) is a re-emerging virus that can cause life-threatening yellow fever disease in humans.Despite the availability of an effective vaccine,little is known about the replication mechanism of YFV,and there are still no available specific anti-YFV medicines.Herein,by introducing the Renilla luciferase gene(Rluc) into an infectious clone of YFV vaccine strain 17 D,we generated a recombinant virus 17 D-Rluc.2 A via reverse genetics approaches.The 17 D-Rluc.2 A had similar plaque morphology and comparable in vitro growth characteristics with its parental strain.Importantly,the reporter luciferase was efficiently expressed in 17 D-Rluc.2 A-infected mammalian and mosquito cells,and there was a good linear correlation between intracellular luciferase expression and extracellular infectious virion reproduction.Furthermore,by a combination of the 17 D-Rluc.2 A reporter virus and selective 2’-hydroxyl acylation analyzed by primer extension(SHAPE)technology,the conserved 5’-SLA element was shown to be essential for YFV replication,highlighting the capability of17 D-R1 uc.2 A in the investigation of YFV replication.At last,we demonstrated that two compounds with distinct anti-viral mechanisms can effectively inhibit the viral propagation in 17 D-Rluc.2 A-infected cells,demonstrating its potential application in the evaluation of anti-viral medicines.Taken together,the 17 D-Rluc.2 A serves as a useful tool for the study of YFV replication and anti-YFV medicine development.
基金partially supported by the National Key R&D Program of China (grant 2018YFC1200602 and 2016YFD0500403 to RHH)。
文摘Zika virus(ZIKV) is associated with severe birth defects and Guillain-Barre′ syndrome and no approved vaccines or specific therapies to combat ZIKV infection are currently available. To accelerate anti-ZIKV therapeutics research, we developed a stable ZIKV GFP-reporter virus system with considerably improved GFP visibility and stability. In this system a BHK-21 cell line expressing DC-SIGNR was established to facilitate the proliferation of GFP-reporter ZIKV. Using this reporter virus system, we established a high-throughput screening assay and screened a selected plant-sourced compounds library for their ability to block ZIKV infection. More than 31 out of 974 tested compounds effectively decreased ZIKV reporter infection. Four selected compounds, homoharringtonine(HHT), bruceine D(BD), dihydroartemisinin(DHA) and digitonin(DGT), were further validated to inhibit wild-type ZIKV infection in cells of BHK-21 and human cell line A549.The FDA-approved chronic myeloid leukemia treatment drug HHT and BD were identified as broad-spectrum flavivirus inhibitors. DHA, another FDA-approved antimalarial drug effectively inhibited ZIKV infection in BHK-21 cells. HHT, BD and DHA inhibited ZIKV infection at a post-entry stage. Digitonin was found to have inhibitory activity in the early stage of viral infection. Our research provides an efficient high-throughput screening assay for ZIKV inhibitors. The active compounds identified in this study represent potential therapies for the treatment of ZIKV infection.
基金This work was supported by the Australian Research Council(ARC)through its Centre of Excellence for Nanoscale BioPhotonics(CE140100003)ARC Discovery Projects(DP200102004).
文摘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.
基金Shanghai Municipal Health and Family Planning Commission Scientific Research Project,Grant/Award Number:20154Y0075Shanghai Municipal Fund for Science and Technology Development,Grant/Award Number:15140904000+1 种基金Shanghai Public Health Clinical Center,Grant/Award Number:KY-GW-2017-04,KY-GW-2018-48National Natural Science Foundation of China,Grant/Award Number:31270217,31601908,81471397
文摘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.
基金financially supported by the National Natural Science Foundation of China (No. 21205094)NFFTBS (Nos. J1103311,J1210057)the New Faculty Startup Funds of Northwest University in Shaanxi Province (No. PR12011)
文摘In this study,a natural cotton thread immunoassay device combined with gold nanorod（GNR） reporter probe is developed for the rapid,sensitive and quantitative electrochemical determination of human ferritin,a lung cancer related biomarker.Human ferritin as an analyte and a pair of monoclonal antibodies are used to demonstrate the proof-of-concept on the cotton thread immunoassay device.An enhancement of the sensitivity is achieved by using gold nanorod as an electroactive report probe compared with a traditional gold nanoparticle（GNP） report probe.The device was capable of measuring 1.58 ng/mL ferritin in 30 min by anodic stripping voltammetry（ASV） testing,which meet the requirement for clinical diagnosis.
