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

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 ...

遺伝学

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 ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

CRISPR/Cas9 dpy-10 Co-CRISPRスクリーニングマーカーと組み立てられたリボヌクレオプロテイン複合体を用いたC.エレガンスrbm-3.2遺伝子の編集。

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

ショウジョウバエのゲノム編集による組織固有のバイナリ転写システムを生成するための効率的な戦略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

発生生物学

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 ...

生物学

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

アスティアナックスメキシコのガメテコレクションと体外受精

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Genome Editing

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of &#8220;genome editing&#8221;, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be introduced into the targeted DNA region. In this video, JoVE explains the principles behind genome editing, emphasizing how this technique relates to DNA repair mechanisms. Then, three major genome editing methods&#8212;zinc finger nucleases, TALENs, and the CRISPR-Cas9 system&#8212;are reviewed, followed by a protocol for using CRISPR to create targeted genetic changes in mammalian cells. Finally, we discuss some current research that applies genome editing to alter the genetic material in model organisms or cultured cells.

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