Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.     

Behead and live long or the tale of cathepsin L

In recent decades Saccharomyces cerevisiae has proven to be one of the most valuable model organisms of aging research. Pathways such as autophagy or the effect of substances like resveratrol and spermidine that prolong the replicative as well as chronological lifespan of cells were described for the first time in S. cerevisiae. In this study we describe the establishment of an aging reporter that allows a reliable and relative quick screening of substances and genes that have an impact on the replicative lifespan. A cDNA library of the flatworm Dugesia tigrina that can be immortalized by beheading was screened using this aging reporter. Of all the flatworm genes, only one could be identified that significantly increased the replicative lifespan of S.cerevisiae. This gene is the cysteine protease cathepsin L that was sequenced for the first time in this study. We were able to show that this protease has the capability to degrade such proteins as the yeast Sup35 protein or the human α‐synuclein protein in yeast cells that are both capable of forming cytosolic toxic aggregates. The degradation of these proteins by cathepsin L prevents the formation of these unfolded protein aggregates and this seems to be responsible for the increase in replicative lifespan.     

Advances in riboswitch-based biosensor as food samples detection tool

Food safety has always been a hot issue of social concern, and biosensing has been widely used in the field of food safety detection. Compared with traditional aptamer-based biosensors, aptamer-based riboswitch biosensing represents higher precision and programmability. A riboswitch is an elegant example of controlling gene expression, where the target is coupled to the aptamer domain, resulting in a conformational change in the downstream expression domain and determining the signal output. Riboswitch-based biosensing can be extensively applied to the portable real-time detection of food samples. The numerous key features of riboswitch-based biosensing emphasize their sustainability, renewable, and testing, which promises to transform engineering applications in the field of food safety. This review covers recent developments in riboswitch-based biosensors. The brief history, definition, and modular design (regulatory mode, reporter, and expression platform) of riboswitch-based biosensors are explained for better insight into the design and construction. We summarize recent advances in various riboswitch-based biosensors involving theophylline, malachite green, tetracycline, neomycin, fluoride, thrombin, naringenin, ciprofloxacin, and paromomycin, aiming to provide general guidance for the design of riboswitch-based biosensors. Finally, the challenges and prospects are also summarized as a way forward stratagem and signs of progress.     

免疫细胞联合腺病毒Ad205-IL-15对肝癌细胞的杀伤研究

在已有腺病毒Ad205-IL-15的基础上尝试与过继免疫细胞CIK、NK92等联合开展对肝癌细胞的杀伤性研究。首次通过荧光化学发光的原理构连出携带表达萤光素酶报告基因的肝癌细胞系Huh7-Luc、MHCC97H—Luc来检测细胞的存活率。结果表明：免疫细胞联合Ad205-IL-15对Huh7-Luc、MHCC97H—Luc两株肝癌细胞表现出了较高协同杀伤作用。证明将免疫细胞与腺病毒联合可以相互弥补各自治疗的不足之处,提高了抗肿瘤效果。同时,由于操作方便,荧光素酶报告基因测定法也为检测细胞存活率提供了新的技术手段。     

AFP与OEA基因的启动子克隆及肿瘤细胞特异性表达的研究

为了研究AFP和CEA基因启：功子的转录活性与肿瘤特异性,本实验利用PCR技术扩增AFP和CEA基因上游序列,并将其克隆到pGL3-Basic载体中构建pGL3-AFP和pGL3-CEA荧光报告质粒。将构建的pGL3-AFP和pGL3-CEA质粒分别与pRL-TK共转染人结肠癌细胞株SW620、肝癌细胞株Huh-7、肺癌细胞株A549、宫颈癌细胞株Hela、人肝正常细胞株QSG7701、人肺正常细胞株Beas-2b,用双荧光报告系统检测这两种质粒在不同细胞里的荧光活性表达。酶切和测序结果证实成功构建了双荧光素酶报告质粒pGL3-AFP和pGL3-CEA,双荧光素酶报告基因检测结果证明AFP和CEA均具有一定的肿瘤特异性。这些结果表明AFP和CEA均具有一定的肿瘤特异性,为进一步研究AFP和CEA基因的表达调控机制和探讨靶向基因-病毒治疗提供依据。     

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.     

Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.     

In situ hybridization of microcolonies using catalyzed reporter deposition with tetramethylbenzidine: a method for detecting low numbers of bacterial cells in drinking water

In this study, catalyzed reporter deposition in situ hybridization (CARD—ISH) with tetramethylbenzidine (TMB) was used for rapid detection of the food pathogens Salmonella typhimurium and Escherichia coli. The bacteria in a sample were concentrated by membrane filtration. The filter membranes with the cells thus removed were incubated on nutrient agar for 4–5 h to allow the formation of microcolonies. Instead of fluorescent tyramide, 3,3′,5,5′-tetramethylbenzidine (TMB), which yields a blue precipitate, was used for signal amplification after in situ hybridization. Microcolonies amplified with TMB produced blue signals, which were sufficiently intense to allow visual evaluation either using a stereomicroscope, or even with the naked eye. Therefore even low cell numbers of hygienically critical bacteria can be detected on the filter membrane without a protracted examination. This enables the detection of low cell numbers (<10 cfu) in a sample of 100 ml tap water within 9–10 h.     

基于.NET的教学质量信息员管理系统分析与设计

教学质量信息员在已成为高校教学质量监控的中坚力量,而良好的信息反馈与沟通方式影响着信息员作用的发挥。信息员提供的信息范围广、数量多导致信息周期长、效率低下,甚至部分信息得不到有效的反馈。该文设计的基于.NET的高校教学质量信息员管理系统将有效地解决这一问题,它具备完善的信息录入、管理、统计、采编和发布等功能。