Name: preparation of raav9 to overexpress or knockdown genes in mouse hearts
Uploaded: 2016-12-17T13:00:00.000+00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

<ol>
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The authors have nothing to disclose.

プラスミド構築の際に望ましくないITR再結合を最小限にすることが重要です。ウイルスを生成する前に、人は必ず制限消化及びアガロースゲル電気泳動を使用してAAVプラスミドのITRの完全性を監視しなければなりません。 100％完全なプラスミドを得ることは不可能であるが、再結合率が可能な限り少なくする必要があります。 20％未満が成功rAAV9包装のために許容可能です。注目すべきは、?...

A correction was made to the Authors section in: <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.
One of the authors names and affiliation was corrected from:
Jian-Ming Jiang3,4 
3 Department of Genetics, Harvard Medical School 
4 Howard Hughes Medical Institute
to:
Jianming Jiang5 
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

A correction was made to the Authors section in: <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.

Erratum: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

様々な生物学的システムにおける特定の遺伝子の発現または活性を制御する遺伝子機能1の研究に貴重な戦略となっています。この目標を達成する直接的な手段は、ヌクレオチド配列を操作し、突然変異対立遺伝子を生成することです。生きた細胞のゲノムに正確な、ターゲットに変更を加えることはまだですが、 時間がかかり、労働集約的な練習、強力なTALENおよびCRISPR / Cas9ツールの開発は、ゲノム編集2-5の新時代を開きました。遺伝子操作のためのより多くのルーチンの実験室方法は、対象1,6の遺伝子（複数可）を発現またはノックダウンするために細胞に遺伝物質（DNAおよびコード配列またはsiRNAを/ shRNAを含有するRNA）の導入に焦点を当ててきました。 多くの場合、遺伝子操作のための主要なボトルネックは、細胞へのDNA、RNA、またはタンパク質の送達です。 in vitro試験、効率的なtransfectiに関してシステム上の多くの培養細胞株で確立されています。しかし、特に、マウスモデルにおいて、 インビボでの遺伝子送達は、より困難です。外因性の試薬の効率的な細胞取り込みを達成するためにバイパスする必要が細胞外および細胞内の障壁のシリーズがあります。追加の障害物は、急速なクリアランスと納入材料7,8の短い期間が含まれます。これらの問題を回避する1つの戦略は、 インビボ遺伝子送達のための「担体」または「ビヒクル」としてウイルスベクターを使用することです。ウイルスの自然に進化した伝達特性は、細胞7,9,10への目的の遺伝子の効率的な送達を可能にします。ウイルスベクターの多くのタイプが開発されたマウスにおける異なる細胞型および器官におけるインビボ遺伝子操作で柔軟可能にされています。 最も一般的に使用されるウイルス系は、レトロウイルス、レンチウイルス、アデノウイルス、およびアデノ随伴ウイルス（AAV）を含みます<s&gt; 11アップ。レトロウイルスは一本鎖RNAウイルスであり、標的細胞および器官12-14における形質導入遺伝子の生涯の発現の可能性を提供し、有糸分裂中に安定的に宿主細胞ゲノムへの遺伝物質を導入することができます。しかし、レトロウイルスの多くのタイプは、分裂細胞に感染し、非分裂細胞におけるそれらの有効性は、15非常に低いです。これは、遺伝子送達のためのそれらの有用性を制限します。レンチウイルスは、 レトロウイルス科の属です。他のレトロウイルスとは異なり、レンチウイルスは、分裂および非分裂細胞の両方に感染することができ、広く有糸分裂後、高度に分化した細胞16への遺伝子導入に使用されています。レンチウイルスのライフサイクルは、宿主ゲノムへのベクターDNAの統合を含みます。このように、レンチウイルス媒介遺伝子送達は、形質導入された遺伝的要素16-18の安定的かつ長期間の発現を可能に。しかし、この機能は、二重Eを表すことができますベクターDNAの統合は、挿入変異誘発につながる可能性として、遺伝子発現を操作するためのこれらのウイルスの使用でdged剣 そして宿主細胞に人為的な影響を引き起こす可能性があります。アデノウイルスは、他の広く使用されている遺伝子送達系です。レトロウイルスおよびレンチウイルスとは異なり、アデノウイルスは、非統合され、宿主細胞8,10,11,19のゲノムの完全性を妨げることはありません。また、アデノウイルスは、多くの細胞型にDNAをトランスフェクトすることができ、感染が活発な細胞分裂19に依存しません。ウイルスベクターは19,20を複製する能力を有するようにアデノウイルスの別の重要な特徴は、ベクター精製の容易さです。しかし、このシステムの主要な警告は、アデノウイルス感染は、特に遺伝子治療の研究において、多くの研究におけるその使用を制限し、標的細胞と臓器19における強力な免疫応答を誘発することができることです。 これらの異なるタイプと比較してウイルスベクターのSは、組換えアデノ随伴ウイルス（rAAV）は、理想的な遺伝子送達系21,22であるように見えます。それは、最小限の免疫原性および病原性23,24を示します。また、rAAVのは、分割および非分裂細胞の両方を含む広範囲の細胞型に感染します。ほとんどの場合、のrAAVは、宿主ゲノムに組み込まれません。従って、標的細胞における望ましくない遺伝子またはゲノムの変化の危険性が低い22です。 最近では、rAAVのシステムが正常マウス心筋23,25-29にタンパク質をコードするDNA、miRNAは、shRNAは、およびCRISPR-gRNAsのインビボ送達のために使用されています。この方法論は、心血管研究の分野における基本的な調査と遺伝子治療の研究を容易にしました。ここでは、詳細な手順を効率的に記述された過剰発現またはマウス心臓における目的の遺伝子をノックダウンrAAV9ベクトルを生成します。プロトコルは、簡単かつ効果的な方法を提供しますマウス実験モデルにおける心臓遺伝子発現を操作します。

<table><tbody><tr><td>Polyethylenimine, Linear (MW 25,000)</td><td>Polysciences, Inc.&amp;nbsp;</td><td>#23966-2</td><td></td></tr><tr><td>Tube, Polypropylene, 36.2 ml, 25 x 87 mm, (qty. 56)</td><td>Beckman Coulter, Inc</td><td># 362183</td><td></td></tr><tr><td>Nuclease, ultrapure</td><td>SIGMA</td><td>#E8263-25KU</td><td></td></tr><tr><td>Density Gradient Medium(Iodixanol)</td><td>SIGMA</td><td>#D1556-250ML</td><td></td></tr><tr><td>Centrifugal Filter Unit with Ultracel-100 membrane</td><td>EMD Millipore Corporation</td><td>#UFC910008</td><td></td></tr><tr><td>Laboratory pipetting needle with 90&amp;deg; blunt ends,gauge 14, L 6 in., nickel plated hub</td><td>SIGMA</td><td>#CAD7942-12EA</td><td></td></tr><tr><td>Poloxamer 188 solution (Pluronic&amp;reg; F-68 solution)</td><td>SIGMA</td><td>P5556-100ML</td><td></td></tr><tr><td>Proteinase K</td><td>SIGMA</td><td>3115828001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Centrifuge machine</td><td>Thermo Scientific</td><td>75004260</td><td></td></tr><tr><td>Centrifuge System</td><td>Beckman Coulter</td><td>363118</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>DMEM medium</td><td>Fisher Scientific</td><td>SH30243FS</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Atlanta Biologicals&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>S11150</td><td></td></tr><tr><td>rAAV9 vector</td><td>Penn Vector Core</td><td>P1967</td><td></td></tr></tbody></table>

preparation of raav9 to overexpress or knockdown genes in mouse hearts

マウスモデルにおける遺伝物質の心筋送達を通して、特定の遺伝子の発現または活性を制御する遺伝子機能の調査を可能にします。心臓におけるそれらの治療可能性を決定することもできます。マウス心臓におけるin vivo分子介入のための限られたアプローチがあります。組換えアデノ随伴ウイルス（rAAV）ベースのゲノム工学は、インビボ心臓遺伝子操作のための不可欠なツールとして利用されています。この技術の具体的な利点は、最小の高効率、高特異性、低ゲノム組込み率を含みます 免疫原性、および最小の病原性。ここでは、パッケージを構築し、rAAV9ベクターを精製するための詳細な手順を説明します。新生児の仔へrAAV9の皮下注射は、肝臓や他の組織に強い発現またはマウス心臓における目的の遺伝子（複数可）の効率的なノックダウンになりますが、ありません。心臓仕を使用しましたC TnnT2プロモーター、心臓におけるGFP遺伝子の高発現が得られました。さらに、rAAV9-U6-shRNAをを利用したときにmRNAが中心部に阻害されたターゲット。 rAAV9技術の知識を作業は、心血管の研究に有用であり得ます。

rAAV9.cTNT :: GFPまたはrAAV9.U6 ::のshRNAプラスミドのrAAV9構築のための戦略は、それぞれ図1及び2に示されています。例として、rAAV9ベクターは、マウス心臓にGFP遺伝子を過剰発現するように生成されました。得られたプラスミドは、2 ITRのサイト（ 図1）によって隣接cTnTの:: GFPカセットを含みます。 rAAV9.U6 :: shRNAベクターがTRBPのmRNAを?...

Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

マウスハーツでの遺伝子を過剰発現またはノックダウンするrAAV9の調製

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

preparation-of-raav9-to-overexpress-or-knockdown-genes-in-mouse-hearts

Universidad San Sebastian

Research

JoVE Journal

Genetics

12.7K ビュー.  Boston Children's Hospital. In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described. 

ビデオ: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

心筋梗塞マウスモデルにおける修飾mRNAの導入

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

遺伝学

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

生物学

エレクトロポレーションに基づくマウス心臓細胞のトランスジェネシス

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

著者スポットライト:迅速な遺伝子スクリーニングのための心臓細胞トランスジェネシス

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

発生生物学

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

心臓の遺伝子がマウスでの編集の CRISPR アデノ随伴ウイルスを介した配信

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

医学

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

マウス胎児心臓の開発に細胞標識と注入

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

GCaMP3レポーターによる心臓特異的カルシウムフラックス測定による心臓リプログラミングのインビトロ評価

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

脈絡叢における選択的遺伝子サイレンシング

Targeted Knockdown of Genes in the Choroid Plexus

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.

著者スポットライト:脈絡叢研究のための遺伝子発現操作技術における洞察と革新 

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

脈絡叢における遺伝子の標的ノックダウン

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

マウスハーツでの遺伝子を過剰発現またはノックダウンするrAAV9の調製

要約

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心筋梗塞マウスモデルにおける修飾mRNAの導入

共焦点イメージングのための成人心筋細胞の単離と遺伝子操作

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

マウス胚性心臓における遺伝子導入のための効率的なトランスジェネシスアプローチ

心臓の遺伝子がマウスでの編集の CRISPR アデノ随伴ウイルスを介した配信

マウス胎児心臓の開発に細胞標識と注入

GCaMP3レポーターによる心臓特異的カルシウムフラックス測定による心臓リプログラミングのインビトロ評価

脈絡叢における遺伝子の標的ノックダウン

脈絡叢における遺伝子の標的ノックダウン