Por favor, ver vídeos relacionados con el derecho <a href="http://jove.com/index/details.stp?ID=718" target="_blank">de los Principios recombinasa específica de sitio (RSS) Tecnología</a> y <a href="http://jove.com/index/details.stp?ID=791" target="_blank">Evolución Molecular de la recombinasa Tre</a> para obtener más información acerca de recombinación específica de sitio y la ingeniería del sitio de la especificidad de las enzimas recombinasa.

The authors have nothing to disclose.

interview: hiv-1 proviral dna excision using an evolved recombinase

VIH-1 se integra en el cromosoma huésped de las células infectadas y persiste como un provirus flanqueado por repeticiones terminales largas. Las actuales estrategias de tratamiento dirigidas principalmente a las enzimas del virus o de fusión virus-célula, la supresión del ciclo de vida viral sin la erradicación de la infección. Dado que el provirus integrado no es víctima de estos enfoques, nuevas cepas resistentes del VIH-1 pueden surgir. Aquí, nos informan de que la ingeniería recombinasa Tre (ver evolución molecular de la recombinasa Tre, Buchholz, F., Instituto Max Planck de Biología Celular y Genética de Dresde) de manera eficiente los impuestos especiales del ADN integrado el VIH-1 proviral en el genoma de las células infectadas. Hemos producido loxLTR contiene pseudotipos viral y las células infectadas HeLa examinar si Tre recombinasa puede extirpar el provirus del genoma del VIH-1 las células humanas infectadas. Un virus de partículas de liberación de la línea celular fue clonado y transfectadas con un plásmido que expresa Tre o con un vector de control parental. Actividad recombinasa y la producción de virus fueron monitoreados. Todos los ensayos demostraron la eliminación eficiente de los provirus de las células infectadas sin visibles efectos citotóxicos. Estos resultados sirven como prueba de principio de que es posible desarrollar una recombinasa para dirigirse específicamente a un LTR del VIH-1 y que esta recombinación es capaz de extirpar el provirus VIH-1 del genoma del VIH-1-las células humanas infectadas. Antes de una recombinación de ingeniería podría entrar en el campo terapéutico, sin embargo, importantes obstáculos que superar. Entre los temas más críticos, los que nos enfrentamos, es una entrega eficiente y segura a las células diana y la ausencia de efectos secundarios.

Watch this Scientific Journal Video about Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase at JoVE.com

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

Actual del VIH-1 estrategias de actuar para suprimir el ciclo de vida del virus, pero no efectiva erradicación de la infección. Aquí, se demuestra que una recombinasa diseñado de manera eficiente los impuestos especiales integrados de VIH-1 proviral del ADN del genoma de las células infectadas.

Entrevista: El VIH-1 La escisión del ADN proviral uso de una recombinasa evolucionado

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

interview-hiv-1-proviral-dna-excision-using-an-evolved-recombinase

Research

JoVE Journal

Biology

11.4K vistas.  Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg. Actual del VIH-1 estrategias de actuar para suprimir el ciclo de vida del virus, pero no efectiva erradicación de la infección. Aquí, se demuestra que una recombinasa diseñado de manera eficiente los impuestos especiales integrados de VIH-1 proviral del ADN del genoma de las células infectadas.

Video: Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: un modelo in vitro para el estudio de la angiogénesis (Entrevista)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Investigación

Biología

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Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Principios de la recombinasa específica de sitio (RSS) Tecnología

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

Entrevista: plegamiento de proteínas y Estudios de Enfermedades Neurodegenerativas

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Evolución molecular de la recombinasa Tre

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Desarrollo de una polimerasa de amplificación ensayo cuantitativo recombinasa con un control positivo interno

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...