この作品はConsejo Nacional de Ciencia yTecnología、CONACYT、México（助成金番号154833）およびNacionalAutónomadeMéxicoの支援を受けました。著者はVíctorGonzález、Rosa I.Santamaría、Patricia Bustos、SoledadJuárezに感謝したい。

<ol>
	<li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77 (1), 61-68 (1989).</li><li>Vos, M. J., Kampinga, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+amplification+strategy+for+unrestricted+generation+of+chimeric+genes.">A PCR amplification strategy for unrestricted generation of chimeric genes.</a> Anal. Biochem. 380 (2), 338-340 (2008).</li><li>Hawkins, N. C., Garriga, G., Beh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+Precise+GFP+Fusions+in+Plasmids+Using+Yeast+Homologous+Recombination.">Creating Precise GFP Fusions in Plasmids Using Yeast Homologous Recombination.</a> Biotechniques. 34 (1), 1-5 (2003).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotechnol. 32 (4), 347-355 (2014).</li><li>Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+recombinase:+The+universal+reagent+for+genome+tailoring.">Cre recombinase: The universal reagent for genome tailoring.</a> Genesis. 26 (2), 99-109 (2000).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Norrander, J., Kempe, T., Messing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+improved+M13+vectors+using+oligodeoxynucleotide-directed+mutagenesis.">Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.</a> Gene. 26 (1), 101-106 (1983).</li><li>Gruber, T. M., Gross, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sigma+subunits+and+the+partitioning+of+bacterial+transcription+space.">Multiple sigma subunits and the partitioning of bacterial transcription space.</a> Annu Rev Microbiol. 57, 441-466 (2003).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Thompson, J. D., Gibson, T. J., Higgins, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sequence+alignment+using+ClustalW+and+ClustalX.">Multiple sequence alignment using ClustalW and ClustalX.</a> Curr Protoc Bioinformatics. , 1-22 (2002).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Cloning:+A+laboratory+manual.">Molecular Cloning: A laboratory manual.</a> CSHLP. , (2001).</li><li>Rutherford, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artemis:+sequence+visualization+and+annotation.">Artemis: sequence visualization and annotation.</a> Bioinformatics. 16 (10), 944-945 (2000).</li></ol>

著者らは競合する金銭的利益がないと宣言している。

CAPRRESIはPRM 2の代替品として設計されました。元のPRMは強力な技術です。それは、選択された遺伝子の任意の部分に沿ったDNA配列の融合を可能にする。 PRMについては、野生型遺伝子を同じプラスミドにクローニングしなければならない。その後、プラスミド上に見出される野生型および抗生物質耐性遺伝子の内部にオリゴヌクレオチドを設計する。プラスミド破壊は、平滑?...

キメラ遺伝子アセンブリは、タンパク質機能を解明するため、および/またはバイオテクノロジー目的のために、分子生物学において広く使用されている。重複するPCR産物増幅1 、プラスミド回収2 、相同組換え3 、CRISPR-Cas9システム4 、部位特異的組換え5 、およびギブソンアセンブリ6のような、遺伝子を融合させるための異なる方法が存在する。これらはそれぞれ異なる技術的利点を提供します。例えば、重複PCR設計の柔軟性、プラスミド回収中の構築物のインビボ選択、またはCRISPR-Cas9およびGibsonシステムの高効率などである。一方、これらの方法のいくつかを実行している間にいくつかの困難が生じることがあります。例えば、最初の2つのアプローチは、平滑末端DNAフラグメントに依存しており、これらのタイプの産物のライゲーションは、sticに比べて技術的に困難であり得るky-endedライゲーション。 部位特異的組換えは、Cre- loxPシステム5のように、オリジナルに余分なDNA配列（傷跡）の痕跡を残す可能性があります。 CRISPR-Cas9は、標的部位4に加えて他のゲノム領域を修飾することがある。 ここでは、プラスミド回収法（PRM）と同義DNA配列に制限酵素部位を挿入し、ライゲーション効率を向上させたタンパク質コード遺伝子を融合させるプロトコールであるプラスミド回収と制限酵素サイト挿入（CAPRRESI）によるキメラアセンブリを紹介する。アミノ酸配列の完全性を保証するために、制限酵素部位を同義語DNA配列のストレッチ上に挿入する。 CAPPRESIの利点の中には、通常の実験用試薬/ツール（ 例えば 、酵素、コンピテントセル、溶液、サーモサイクラー）を使用して行うことができ、迅速な結果を得ることができるという点が挙げられます。制限に依拠する同義語DNA配列から出現する酵素部位は、目的のタンパク質内の正確な融合点の選択を制限し得る。そのような場合、重複するオリゴヌクレオチドを用いて標的遺伝子を融合させ、制限酵素部位をベクターの耐性遺伝子上に挿入しなければならない。 CAPRRESIは、7つの簡単なステップ（ 図1 ）からなる：1）クローニングベクターpUC18またはpUC19 6の選択 ; 2）融合される野生型配列のin silico分析; 3）キメラアセンブリおよびプラスミド破壊のための破壊領域の選択; 4）制限酵素部位を含む同義語DNA配列のin silico生成; 5）選択されたプラスミドへの野生型遺伝子の独立したクローニング; 6）PCRによるプラスミド破壊、続いて制限酵素消化;および7）DNA連結および細菌形質転換を用いたプラスミド回収。この技術によって産生されるキメラ遺伝子は、i番目のシーケンス。 pUC18 / 19ベクターは、小型（ すなわち、 2,686塩基対）、高コピー数、有利な配列決定反応特徴および商業的利用可能性などのクローニングおよびキメラアセンブリーに技術的利点をもたらす。ここでは、 大腸菌（Escherichia coli）宿主を用いて、バクテリアの培養物が安価で増殖が速いため、キメラを組み立てて取り扱う。このことを考慮して、最終標的プラスミドへの融合断片のその後のクローニングが必要となる（ 例えば、細菌におけるpRK415としての発現ベクターまたは哺乳類細胞におけるpCMVとしての発現ベクター）。 CAPRRESIは、2つの主要なシグマ因子遺伝子、 E. colirpoDおよびRhizobium etlisigAを融合させるために試験された。一次シグマ因子は、転写開始に関与するRNAポリメラーゼサブユニットであり、4つのドメイン（ すなわち、 σ1、σ2、σ3、およびσ4） 8からなる 。アミノ酸配列の長さrpoDおよびsigAによってコードされるタンパク質はそれぞれ613および685である。 RpoDとSigAは48％の同一性（98％のカバレッジ）を共有します。これらの一次シグマ因子は、領域σ2とσ3との間で2つの相補的フラグメントに分割された。キメラ01（RpoDσ1-σ2+SigAσ3-σ4）およびキメラ02（SigAσ1-σ2+RpoDσ3-σ4）の2つのキメラ遺伝子をこのデザインに従って組み立てた。 DNA融合産物は、配列決定によって確認した。

<table><tbody><tr><td>Platinum Taq DNA polymerase High Fidelity</td><td>Thermo Fisher</td><td>11304011</td><td>Produces a mix of blunt/3&amp;rsquo;-A overhang ended PCR products</td></tr><tr><td>High pure plasmid isolation kit</td><td>Roche</td><td>11754785001</td><td>Used for all plasmid purification reactions</td></tr><tr><td>High pure PCR product purification kit</td><td>Roche</td><td>11732676001</td><td>Used for all PCR purification reactions from agarose gels</td></tr><tr><td>AflII restriction enzyme</td><td>New England Biolabs</td><td>R0520L</td><td>Recognizes sequence 5&amp;rsquo;-CTTAAG-3&amp;rsquo; and cuts at 37 &amp;deg;C</td></tr><tr><td>KpnI-HF restriction enzyme</td><td>New England Biolabs</td><td>R3142L</td><td>Recognizes sequence 5&amp;rsquo;-GGTACC-3&amp;rsquo; and cuts at 37 &amp;deg;C</td></tr><tr><td>SpeI-HF restriction enzyme</td><td>New England Biolabs</td><td>R3133L</td><td>Recognizes sequence 5&amp;rsquo;-ACTAGT-3&amp;rsquo; and cuts at 37 &amp;deg;C</td></tr><tr><td>XbaI restriction enzyme</td><td>New England Biolabs</td><td>R0145L</td><td>Recognizes sequence 5&amp;rsquo;-TCTAGA-3&amp;rsquo; and cuts at 37 &amp;deg;C</td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A0166-5G</td><td>Antibiotics</td></tr><tr><td>Nalidixic acid</td><td>Sigma Aldrich</td><td>N8878-5G</td><td>Antibiotics</td></tr><tr><td>Yeast Extract</td><td>Sigma Aldrich</td><td>Y1625-1KG</td><td>Bacterial cell culture</td></tr><tr><td>Casein peptone</td><td>Sigma Aldrich</td><td>70171-500G</td><td>Bacterial cell culture</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S9888-1KG</td><td>Sodium chloride</td></tr><tr><td>Agar</td><td>Sigma Aldrich</td><td>05040-1KG</td><td>Bacterial cell culture</td></tr><tr><td>&lt;em&gt;XbaI&lt;/em&gt; restriction enzyme</td><td>Thermo Fisher</td><td>FD0684</td><td>Fast digest &lt;em&gt;XbaI&lt;/em&gt; enzyme</td></tr><tr><td>&lt;em&gt;KpnI&lt;/em&gt; restriction enzyme</td><td>Thermo Fisher</td><td>FD0524</td><td>Fast digest &lt;em&gt;KpnI &lt;/em&gt;enzyme</td></tr><tr><td>Quick Ligation Kit</td><td>New England Biolabs</td><td>M2200S</td><td>Fast DNA ligation kit</td></tr><tr><td>&lt;em&gt;AflII&lt;/em&gt; restriction enzyme</td><td>Thermo Fisher</td><td>FD0834</td><td>Fast digest &lt;em&gt;AflII &lt;/em&gt;enzyme</td></tr><tr><td>&lt;em&gt;SpeI&lt;/em&gt; restriction enzyme</td><td>Thermo Fisher</td><td>FD1253</td><td>Fast digest &lt;em&gt;SpeI &lt;/em&gt;enzyme</td></tr></tbody></table>

caprresi: chimera assembly by plasmid recovery and restriction enzyme site insertion

ここでは、プラスミド回収および制限酵素部位挿入（CAPRRESI）によるキメラアセンブリを提示する。 CAPRRESIは、元のプラスミド回収法の多くの利点から利益を得、制限酵素消化を導入してDNAライゲーション反応を容易にする（キメラアセンブリに必要とされる）。このプロトコルでは、ユーザーは野生型遺伝子を同じプラスミド（pUC18またはpUC19）にクローニングします。キメラが組み立てられるべきアミノ酸配列領域のin silico選択の後、ユーザーはそれらをコードするすべての同義語DNA配列を得る。 Ad Hoc Perlスクリプトを使用すると、ユーザーはすべてのシノニムDNA配列を決定できます。このステップの後、別のPerlスクリプトは、すべての同義語DNA配列上の制限酵素部位を検索する。このin silico分析はまた、pUC18 / 19プラスミド上に見出されるアンピシリン耐性遺伝子（ ampR ）を用いて行われる。ユーザは、野生型およびampR遺伝子をPCRによって破壊するために同義語領域内のオリゴヌクレオチドを設計する。 obtの後相補的なDNA断片を精製し、精製し、制限酵素消化を行う。適切な相補的DNA断片を連結することにより、キメラ構築が達成される。 CAPRRESIのために、小さいサイズ（2,686塩基対）、高いコピー数、有利な配列決定反応特徴および商業的入手可能性などの技術的利点を提供するため、pUC18 / 19ベクターが選択される。キメラ構築のための制限酵素の使用は、平滑末端生成物を産生するDNAポリメラーゼの必要性を排除する。 CAPRRESIは、タンパク質コード遺伝子を融合するための迅速かつ低コストの方法である。

図1に CAPRRESIを示します。この方法を用いて、2つの細菌の一次シグマ因子（ すなわち、大腸菌 RpoDおよびR. etli SigA）のドメインを交換することによって、2つのキメラ遺伝子を組み立てた。 rpoDおよびsigA遺伝子のDNA配列は、それぞれGenBankゲノムファイルNC_000913およびNC_007761からArtemis Genome Browser 14を使用して得?...

Watch this Scientific Journal Video about CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion at JoVE.com

CAPRRESI: プラスミド回収と制限酵素部位挿入によるキメラアセンブリ |生物学 |ビデオ

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

ここでは、同義語DNA配列への制限酵素部位の挿入および機能的プラスミド回収に基づくプロトコルであるプラスミド回収および制限酵素部位挿入（CAPRRESI）によるキメラアセンブリを提示する。このプロトコールは、タンパク質コード遺伝子を融合するための迅速かつ低コストの方法である。

CAPRRESI：プラスミド回収および制限酵素部位挿入によるキメラアセンブリ

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...

caprresi-chimera-assembly-plasmid-recovery-restriction-enzyme-site

Universidad de Cartagena UDEC

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SCIENTISTS IN ACTION

11.5K ビュー.  Universidad Nacional Autónoma de México. ここでは、同義語DNA配列への制限酵素部位の挿入および機能的プラスミド回収に基づくプロトコルであるプラスミド回収および制限酵素部位挿入（CAPRRESI）によるキメラアセンブリを提示する。このプロトコールは、タンパク質コード遺伝子を融合するための迅速かつ低コストの方法である。 

ビデオ: CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

キメラ蛋白質の構築による機能性タンパク質領域の同定

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

生化学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

全プラスミドの自家製のサイトが特異的突然変異誘発

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

生物学

<meta charset="utf8"></head><body>分子生物学の教育とスキルを強化するためのGibson Assemblyの実装

Improving Student Outcomes with an Adaptable Molecular Cloning Course-Based Undergraduate Research Experience

The continuous advancement of molecular biology techniques requires that molecular biology curricula are&#160;regularly refined to effectively prepare students to enter the workforce with modern competencies. In particular, the emergence of Gibson Assembly, a highly customizable and adaptive molecular cloning technique, has advanced the landscape of molecular cloning in numerous research environments. Thus, we created a Gibson Assembly cloning module for deployment in a molecular biology laboratory course at California Polytechnic State University, San Luis Obispo and evaluated student learning outcomes from the module. Over three iterations of the course, students participated in an experiment-based independent project that involved cloning three unique plasmid libraries to support research projects in natural products biosynthesis. Students were given pre- and post-questionnaires to evaluate their understanding of molecular cloning and their confidence in molecular biology terms and techniques. Students&#8217; responses showed a significant increase in both learning molecular cloning concepts and in self-reported confidence with molecular cloning terms and techniques. This module framework can be generalized to teach Gibson Assembly for various applications, providing instructors with a toolkit for teaching an adaptable and emergent cloning technology while advancing their research projects.

<meta charset="utf8"></head><body>適応性のある分子クローニングコースベースの学部研究経験による学生の成果の向上

An adaptable Gibson Assembly molecular cloning module was employed in a course-based undergraduate research experience (CURE) format for molecular biology laboratory course students. Assessment of student learning outcomes showed improved understanding and confidence in molecular cloning after completion of the CURE, and novel plasmids were cloned for natural product biosynthesis research.&#160;&#160;

適応性のある分子クローニングコースベースの学部研究経験による学生の成果の向上

The continuous advancement of molecular biology techniques requires that molecular biology curricula are&#160;regularly refined to effectively prepare ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

生物工学

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

クラミジア・トラコマティスにおけるフロキセカセットアレリック交換変異誘発によるマーカーレス遺伝子欠失

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

免疫・感染学

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

高効率プラスミド構築のための In vivo アセンブリの利用

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

サブクローニングプラス挿入（SPI） - ジーンターゲティングベクターの迅速な構築のための新しい遺伝子改変方法

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

CAPRRESI：プラスミド回収および制限酵素部位挿入によるキメラアセンブリ

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キメラ蛋白質の構築による機能性タンパク質領域の同定

全プラスミドの自家製のサイトが特異的突然変異誘発

適応性のある分子クローニングコースベースの学部研究経験による学生の成果の向上

適応性のある分子クローニングコースベースの学部研究経験による学生の成果の向上

適応性のある分子クローニングコースベースの学部研究経験による学生の成果の向上

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

クラミジア・トラコマティスにおけるフロキセカセットアレリック交換変異誘発によるマーカーレス遺伝子欠失

Construction of Adenoviral Vectors using DNA Assembly Technology

高効率プラスミド構築のための In vivo アセンブリの利用

サブクローニングプラス挿入（SPI） - ジーンターゲティングベクターの迅速な構築のための新しい遺伝子改変方法