と題する関連動画を表示してください<a href="http://jove.com/index/details.stp?ID=718" target="_blank">部位特異的リコンビナーゼ（SSR）技術の原理</a>と<a href="http://jove.com/index/details.stp?ID=791" target="_blank">トレリコンビナーゼの分子進化</a>部位特異的組換えとエンジニアリングリコンビナーゼ酵素の部位特異性の詳細については、を。

The authors have nothing to disclose.

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

HIV - 1は感染細胞の宿主染色体に統合し、長い末端反復配列に挟まれたプロウイルスとして持続します。現在の治療戦略は、主に感染症を根絶することなく、ウイルスの酵素やウイルスの細胞融合、抑制するウイルスのライフサイクルを対象としています。統合されたプロウイルスは、これらの手法によって対象とされていないため、HIV - 1の新たな耐性菌が出現する可能性があります。ここで、我々は人工リコンビナーゼトレは（トレリコンビナーゼ、バックホルツ、F.、細胞生物学および遺伝学のマックスプランク研究所、ドレスデンの分子進化を参照）効率的に感染細胞のゲノムから統合されたHIV - 1プロウイルスDNAを消費税と報告している。我々は、トレリコンビナーゼは、HIV - 1感染ヒト細胞のゲノムから切り出すプロウイルスをできるかどうかを調べるためにloxLTR含むウイルス疑似型と感染したHeLa細胞を作り出した。ウイルス粒子放出細胞株をクローニングし、発現プラスミドトレでまたはペアレンタルコントロールのベクターでトランスフェクトした。リコンビナーゼ活性とウイルス産生をモニターした。すべてのアッセイは、目に見える細胞傷害作用することなく感染細胞からプロウイルスの効率的な削除を示した。これらの結果は、それが具体的にHIV - 1 LTRをターゲットにリコンビナーゼを進化させることが可能であり、このリコンビナーゼは、HIV - 1感染ヒト細胞のゲノムからHIV - 1プロウイルスを切り出すことが可能であることを原理の証明として役立つ。 設計リコンビナーゼは、治療の分野に入ることができる前に、しかし、重大な障害が克服される必要があります。最も重要な問題の中で、我々が直面している、標的細胞への効率的かつ安全に配信して副作用のないことです。

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

現在のHIV - 1の戦略は、ウイルスのライフサイクルを抑制する働きが効果的に感染を根絶することはありません。ここで、我々は人工リコンビナーゼは、感染細胞のゲノムから効率的に切り出す統合されたHIV - 1プロウイルスDNAができることを示している。

インタビュー：進化リコンビナーゼを用いたHIV - 1プロウイルスDNAの切り出し

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

11.3K ビュー.  Heinrich-Pette-Institute for Experimental Virology and Immunology, University of Hamburg. 現在のHIV - 1の戦略は、ウイルスのライフサイクルを抑制する働きが効果的に感染を根絶することはありません。ここで、我々は人工リコンビナーゼは、感染細胞のゲノムから効率的に切り出す統合されたHIV - 1プロウイルスDNAができることを示している。

ビデオ: 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.

クリストファーヒューズ：血管新生の研究のためのin vitroモデル（インタビュー）

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

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.

インタビュー - シロアリ後腸内微生物群集の調査

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

自然と実験室での微生物群集 - インタビュー

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

マイクロ流体を用いた細菌走化性の研究 - インタビュー

Biology of Microbial Communities  - Interview

微生物群集の生物学 - インタビュー

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

コレラ菌：細菌性病因を研究するモデル生物 - インタビュー

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.

部位特異的リコンビナーゼ（SSR）技術の原理

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.

インタビュー：タンパク質フォールディングと神経変性疾患の研究

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.

トレリコンビナーゼの分子進化

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.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

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