관련 동영상받을 참조하십시오 <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=718" target="_blank">특정 사이트 Recombinase (SSR) 기술의 원리</a> 와 <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=791" target="_blank">트레의 Recombinase의 분자 진화를</a> 특정 사이트 재조합 및 엔지니어링 recombinase 효소의 사이트 특이성에 대한 자세한 내용은.

The authors have nothing to disclose.

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

HIV - 1 감염 세포의 호스트 염색체에 통합과 긴 터미널 반복 둘러싸인 provirus로 지속. 현재 치료 전략은 주로 감염을 근절하지 않고 바이러스 효소 또는 바이러스 - 세포 융합, 억제 바이러스 생활주기를 대상으로. 통합 provirus가 이러한 방식을, HIV - 1의 새로운 내성 변종의 대상이 아니므로 등장 수도 있습니다. 여기서 우리는 설계 recombinase 트레가 (트레 recombinase, 뷰큘츠, F., 세포 생물학 및 유전학에 대한 최대 플랑크 연구소, 드레스덴의 분자 진화를 참조) 효율적으로 감염 세포의 게놈에서 통합 HIV - 1 proviral DNA를 excises 것을보고합니다. 우리는 바이러스 검사 및 감염 pseudotypes 헬라 세포를 포함하는 loxLTR을 생산 트레 recombinase는 HIV - 1 감염 인간 세포의 게놈에서 소비세 provirus 수 있는지 여부. 바이러스 입자 - 방출 셀 라인은 복제와 플라스미드 표현 트레 또는 보호자 제어 벡터와 transfected했다. Recombinase 활동 및 바이러스 생산을 감시했다. 모든 assays 보이는 세포 독성 효과없이 감염된 세포에서 provirus의 효율적인 삭제를 보여주었다. 이러한 결과는 특히 HIV - 1 LTR를 대상으로 recombinase를 발전 가능이 recombinase는 HIV - 1에 감염된 인간 세포의 게놈에서 HIV - 1 provirus을 절개 수있다는 것을 원칙의 증거로 제공하고 있습니다. 설계 recombinase가 치료 올라와있을 전에 그러나, 큰 장애물은 극복해야합니다. 가장 중요한 문제 중 우리가 얼굴 즉, 타겟 세포에 효율적이고 안전하게 전달 및 부작용의 부재입니다.

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

현재 HIV - 1 전략은 바이러스 생활주기를 억제하지만, 효과적으로 감염을 근절하지 행동. 여기, 우리는 입증되는 감염된 세포의 게놈에서 설계 recombinase 수 효율적으로 소비세 통합 HIV - 1 proviral DNA.

인터뷰 : 진화 Recombinase을 사용하여 HIV - 1의 DNA 절단 Proviral

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 조회수.  Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg. 현재 HIV - 1 전략은 바이러스 생활주기를 억제하지만, 효과적으로 감염을 근절하지 행동. 여기, 우리는 입증되는 감염된 세포의 게놈에서 설계 recombinase 수 효율적으로 소비세 통합 HIV - 1 proviral DNA.

비디오: 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.

크리스토퍼 휴즈 : Angiogenesis 연구를위한 체외 모델 (인터뷰)

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

연구

생물학

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.

인터뷰 - 마이트의 H​​indgut에서 미생물 커뮤니티를 조사

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

자연과 실험실에서 미생물 커뮤니티 - 인터뷰

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

Microfluidics를 사용하여 세균 Chemotaxis의 연구 - 인터뷰

Biology of Microbial Communities  - Interview

미생물 커뮤니티의 생물학 - 인터뷰

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

비브리오 cholerae : 박테리아 Pathogenesis을 공부 모델 생물체 - 인터뷰

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.

특정 사이트 Recombinase (SSR) 기술의 원리

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.

인터뷰 : 단백질 폴딩과 Neurodegenerative 질병의 연구

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.

트레 Recombinase의 분자 진화

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.