この作品は、遺伝学、開発および細胞生物学、アイオワ州立大学の学科によっておよびNIHのグラントEY024941（WJ）.DRによって賄われていました。ジェフリーEssnerは、原稿にコメントを提供しました。

The authors have nothing to disclose.

長足の進歩は、形質の進化の遺伝的基礎を理解することに向けて、近年で行われています。特徴の数の進化の基礎となる候補遺伝子が同定されたが、それが原因で最も進化興味深い種の遺伝的取り扱いやすさの欠如にインビボでこれらの遺伝子を試験するために困難なままです。ここでは、Aのゲノム編集のための方法を報告しますmexicanus、洞窟動物の進化を研究するため...

形質進化の遺伝的基礎を理解することは、進化の生物学者の重要な研究目標です。かなりの進歩は、形質の発生の根底にある遺伝子座を同定し、（実施例1-3の場合）、これらの遺伝子座内の候補遺伝子を特定してなされました。形質の進化を研究するために使用される多くの生物は現在、遺伝的に扱いやすいではありませんしかし、機能的にこれらの遺伝子の役割をテストすることは困難なままです。ゲノム編集技術の出現が大幅に広範囲の生物の遺伝的操作性を増加しています。転写活性化因子のようなエフェクターヌクレアーゼ（TALENs）とクラスタ化された定期的interspaced短いパリンドロームリピート（CRISPRs）は、（例えば、4月11日のために）多数の生物の遺伝子の標的に変異を生成するために使用されてきました。進化的に関連したシステムに適用されるこれらのツールは、進化生物学者は進化の遺伝的基礎を勉強方法に革命をもたらす可能性を秘めています。 アステュアナクスのmexicanusは、2つの形態で存在する魚の種である：川に生息する表面形状（表面の魚）と複数の洞窟住居形態（洞窟魚）A. mexicanusの洞窟魚は（12に概説されている）表面の魚の祖先から進化しました。洞窟魚の集団味蕾と頭蓋neuromastsの数を増加させ、色素沈着の減少または損失を目の損失を含む形質の数を進化させてきた、そのような学校教育の行動の損失のような行動の変化は、給紙姿勢と過食症13の変化を侵略を増加しました-19。洞窟魚と表面の魚は異系交配できるであり、遺伝的マッピング実験は、洞窟の形質1,20-26のための遺伝子座および候補遺伝子を同定するために行われてきました。いくつかの候補遺伝子は、他の種21または過剰発現27または過渡ノックダウンUでのモデル生物で、細胞培養1,19で洞窟の特性に貢献する機能的役割のためにテストされていますA.でモルフォリノ28を歌いますmexicanus。しかしながら、これらの方法の各々には限界があります。 A.これらの遺伝子の変異対立遺伝子を生成する能力mexicanusは洞窟魚の進化にそれらの機能を理解するために重要です。したがって、A. mexicanusは、ゲノム編集技術の応用のための理想的な候補生物です。 ここでは、Aのゲノム編集のための方法を概説しますmexicanus TALENsを使用。この方法は、表現型および興味29の遺伝子で安定突然変異を有する魚の行を単離するためのモザイク注入創始魚を評価するために使用することができます。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td></td><td></td><td></td></tr><tr><td>Injection station</td><td></td><td></td><td></td></tr><tr><td>Gel apparatus</td><td></td><td></td><td></td></tr><tr><td>Needle puller</td><td></td><td></td><td></td></tr><tr><td>Nanodrop</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Note: As far as we know, supplies from different companies can be used unless otherwise indicated</td><td></td><td></td><td></td></tr><tr><td>Golden Gate TALEN and TAL Effector Kit 2.0</td><td>Addgene</td><td>Kit #1000000024</td><td></td></tr><tr><td>Fisher BioReagents LB Agar, Miller (Granulated)</td><td>Fisher</td><td>BP9724-500</td><td></td></tr><tr><td>Fisher BioReagents Microbiology Media: LB Broth, Miller</td><td>Fisher</td><td>BP1426-500</td><td></td></tr><tr><td>Teknova TET-15 in 50% EtOH</td><td>Teknova (ordered through Fisher)</td><td>50-843-314</td><td></td></tr><tr><td>Spectinomycin Dihydrochloride, Fisher BioReagents</td><td>Fisher</td><td>BP2957-1</td><td></td></tr><tr><td>Super Ampicillin (1,000x solution)</td><td>DNA Technologies</td><td>6060-1</td><td></td></tr><tr><td>ThermoScientific X-Gal Solution, ready-to-use</td><td>Thermo Sci Fermentas Inc (Ordered through Fisher)</td><td>FERR0941</td><td></td></tr><tr><td>IPTG, Fisher BioReagents</td><td>Fisher</td><td>BP1620-1</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher</td><td>08-757-13</td><td></td></tr><tr><td>&lt;em&gt;Bsa&lt;/em&gt;I</td><td>New England Biolabs (ordered through Fisher)</td><td>50-812-203</td><td>Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td></tr><tr><td>BSA</td><td>New England Biolabs</td><td>provided with restriction enzymes</td><td></td></tr><tr><td>10x T4 ligase buffer</td><td>Promega (ordered through Fisher)</td><td>PR-C1263</td><td></td></tr><tr><td>GoTaq Green Master mix</td><td>Promega (ordered through Fisher)</td><td>PRM7123</td><td>Other Taq can be used, but the reaction should be adjusted accordingly</td></tr><tr><td>Quick ligation kit</td><td>New England Biolabs (ordered through Fisher)</td><td>50-811-728</td><td>We use Quick Ligase for all TALEN assembly reactions</td></tr><tr><td>One Shot TOP10 Chemically Competent E.coli</td><td>Invitrogen</td><td>C4040-06</td><td>Other chemically competent cells or homemade competent cells can be used</td></tr><tr><td>&lt;em&gt;Esp&lt;/em&gt; 3I</td><td>Thermo Sci Fermentas Inc (Ordered through Fisher)</td><td>FERER0451</td><td></td></tr><tr><td>Plasmid-Safe ATP-dependent DNase</td><td>Epicentre (Ordered through Fisher)</td><td>NC9046399</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>The Qiagen kit should be used for the initial plasmid preparation (as described in the Golden Gate TALEN and TAL Effector Kit protocol)</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>GeneMate LE Quick Dissolve Agaraose</td><td>BioExpress</td><td>E-3119-125</td><td></td></tr><tr><td>&lt;em&gt;Sac&lt;/em&gt; I</td><td>Promega (Ordered through Fisher)</td><td>PR-R6061</td><td></td></tr><tr><td>mMESSAGE mMACHINE T3 Transcription kit</td><td>Ambion</td><td>AM1348M</td><td></td></tr><tr><td>Rneasy MinElute Cleanup Kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>NorthernMax-Gly Sample Loading Dye&amp;nbsp;</td><td>Ambion (ordered through Fisher)</td><td>AM8551</td><td></td></tr><tr><td>Eliminase</td><td>Decon (ordered through Fisher)</td><td>04-355-32</td><td></td></tr><tr><td>Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets</td><td>Fisher</td><td>13-678-6B</td><td></td></tr><tr><td>Standard Glass Capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td></td></tr><tr><td>Microcaps</td><td>Drummond Scientific Company</td><td>1-000-0010</td><td></td></tr><tr><td>Eppendorf Femtotips Microloader Tips for Femtojet Microinjector</td><td>Eppendorf (ordered through Fisher)</td><td>E5242956003</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher</td><td>S318-500</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP152-1</td><td></td></tr></tbody></table>

genome editing in astyanax mexicanus using transcription activator-like effector nucleases (talens)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

TALENペア注射は、非相同末端結合（NHEJ）を介して修復することができる二本鎖休憩39で、その結果、特定のDNAヌクレオチドとRVDSの結合、したがってのFokIドメインの二量体化につながります。 NHEJは、多くの場合、挿入または欠失（インデル）につながるエラーを導入しています。インデルはTALEN標的部位を囲む領域を増幅しTALENスペーサ領域内で切断す...

Watch this Scientific Journal Video about Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs at JoVE.com

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

遺伝子標的化突然変異誘発は、ゲノム編集の技術を使用して、生物の幅広いことが可能です。ここで、我々はアステュアナクスmexicanus、表面の魚や洞窟魚が含まれて魚の種におけるエフェクターヌクレアーゼ（TALENs）などの転写活性化因子を用いて標的遺伝子の突然変異誘発のためのプロトコルを示しています。

ゲノム編集中<em&gt;アステュアナクスmexicanus</em&gt;使用して転写活性化因子のようなエフェクターヌクレアーゼ（TALENs）

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

genome-editing-astyanax-mexicanus-using-transcription-activator-like

Research

JoVE Journal

Biology

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs)

8.2K ビュー.  Iowa State University. 遺伝子標的化突然変異誘発は、ゲノム編集の技術を使用して、生物の幅広いことが可能です。ここで、我々はアステュアナクスmexicanus、表面の魚や洞窟魚が含まれて魚の種におけるエフェクターヌクレアーゼ（TALENs）などの転写活性化因子を用いて標的遺伝子の突然変異誘発のためのプロトコルを示しています。 

ビデオ: Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

遺伝学

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

効率的な生産とモデル システムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

強化されたゲノム細胞や生物の多様な Cas9 リボ核蛋白質と編集

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

発生生物学

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

Post-larval 表現型解析およびホール マウント免疫組織化学のためメキシコ テトラアステュアナクス メキシコの調達

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

メキシコの血脈遺伝子機能の操作

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.

Presented here is a protocol to perform time- and space-restricted gene knock-out in axolotl spinal cords by injecting CAS9-gRNA complex into the spinal cord central canal followed by electroporation.

CAS9タンパク質-gRNA複合体のエレクトロポレーションによるアクソロトル脊髄神経幹細胞の直接遺伝子ノックアウト

The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

デザイナーヌクレアーゼを用いてマウスゲノムエンジニアリング

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

ゲノム編集中

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アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

効率的な生産とモデルシステムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

強化されたゲノム細胞や生物の多様な Cas9 リボ核蛋白質と編集

Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

Post-larval 表現型解析およびホールマウント免疫組織化学のためメキシコテトラアステュアナクスメキシコの調達

メキシコの血脈遺伝子機能の操作

CAS9タンパク質-gRNA複合体のエレクトロポレーションによるアクソロトル脊髄神経幹細胞の直接遺伝子ノックアウト

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

デザイナーヌクレアーゼを用いてマウスゲノムエンジニアリング

ゲノム編集中

要約

さらに動画を探す

この動画の章

アフリカツメガエルのリープフロッギング CRISPR/Cas9 ベースの始原生殖細胞の移植

効率的な生産とモデル システムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

強化されたゲノム細胞や生物の多様な Cas9 リボ核蛋白質と編集

Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

Post-larval 表現型解析およびホール マウント免疫組織化学のためメキシコ テトラアステュアナクス メキシコの調達

メキシコの血脈遺伝子機能の操作

CAS9タンパク質-gRNA複合体のエレクトロポレーションによるアクソロトル脊髄神経幹細胞の直接遺伝子ノックアウト

ヒト多能性幹細胞におけるCRISPR-CAS9を用いて定義されたゲノム修飾の生成

In Situ ハイブリダイゼーションアステュアナクス胚の Wholemount

デザイナーヌクレアーゼを用いてマウスゲノムエンジニアリング

効率的な生産とモデルシステムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

Post-larval 表現型解析およびホールマウント免疫組織化学のためメキシコテトラアステュアナクスメキシコの調達