请查看相关影片题为<a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=718" target="_blank">“网站具体的重组酶（SSR）</a>技术<a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=791" target="_blank">的原理和TRE重组</a>酶的分子进化位点特异性重组和工程站点特异性重组酶的有关信息。

The authors have nothing to disclose.

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

HIV - 1感染细胞整合到宿主染色体和一个由两侧的长末端重复序列的前病毒仍然存在。目前的治疗策略主要目标没有消除感染病毒酶或病毒细胞融合，抑制病毒的生命周期。由于整合前病毒是不通过这些方法，新的耐药菌株的HIV - 1的目标，可能会出现。在这里，我们报告，工程重组TRE（TRE重组，巴克霍尔兹，楼，细胞生物学和遗传学的马克斯普朗克研究所，德累斯顿的分子进化）有效地切除感染细胞的基因组整合的HIV - 1前病毒DNA。 TRE重组是否可以消费的前病毒HIV - 1感染人体细胞的基因组，我们生产的loxLTR含有伪病毒感染的HeLa细胞研究。病毒粒子释放细胞株克隆转染与表达质粒TRE或家长控制向量。重组酶的活性和病毒的生产进行了监测。所有实验证明，无可见的细胞毒作用的前病毒从被感染的细胞的有效删除。这些结果作为证据原则，有可能进化出专门针对HIV - 1 LTR的重组，这种重组是能够切除的HIV - 1前病毒HIV - 1感染人体细胞的基因组。 工程重组之前可以进入的治疗上，然而，重大的障碍需要克服。其中最关键的问题，是我们面临的，是一种高效，安全的运送到靶细胞，并没有副作用。

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

现有的HIV - 1战略采取行动来抑制病毒的生命周期，但不能有效地消除感染。在这里，我们证明了一个精心设计的重组可以有效地消费综合HIV - 1前病毒从感染细胞的基因组DNA。

专访：HIV - 1前病毒DNA切除使用一个演进的重组酶

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

Nanyang Technological University

Research

JoVE Journal

Biology

11.4K 次观看.  Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg. 现有的HIV - 1战略采取行动来抑制病毒的生命周期，但不能有效地消除感染。在这里，我们证明了一个精心设计的重组可以有效地消费综合HIV - 1前病毒从感染细胞的基因组DNA。

视频: 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.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

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

科研

生物学

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

调查白蚁后肠中的微生物的社区 - 面试

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

使用微流体的细菌的趋化作用的研究 - 访

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

霍乱弧菌：模式生物研究细菌的发病机制 - 访

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.

特定地点的重组酶（SSR）技术的原则

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.

专访：蛋白质折叠与神经退行性疾病的研究

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.

TRE重组酶的分子进化

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

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.