ゲノム編集中<em>アステュアナクスmexicanus</em>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）
Summary
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。
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Equipment | |||
| Thermocycler | |||
| Injection station | |||
| Gel apparatus | |||
| Needle puller | |||
| Nanodrop | |||
| Name | Company | Catalog Number | Comments |
| Supplies | |||
| Note: As far as we know, supplies from different companies can be used unless otherwise indicated | |||
| Golden Gate TALEN and TAL Effector Kit 2.0 | Addgene | Kit #1000000024 | |
| Fisher BioReagents LB Agar, Miller (Granulated) | Fisher | BP9724-500 | |
| Fisher BioReagents Microbiology Media: LB Broth, Miller | Fisher | BP1426-500 | |
| Teknova TET-15 in 50% EtOH | Teknova (ordered through Fisher) | 50-843-314 | |
| Spectinomycin Dihydrochloride, Fisher BioReagents | Fisher | BP2957-1 | |
| Super Ampicillin (1000x solution) | DNA Technologies | 6060-1 | |
| ThermoScientific X-Gal Solution, ready-to-use | Thermo Sci Fermentas Inc (Ordered through Fisher) | FERR0941 | |
| IPTG, Fisher BioReagents | Fisher | BP1620-1 | |
| Petri dishes | Fisher | 08-757-13 | |
| BsaI | New England Biolabs (ordered through Fisher) | 50-812-203 | Use BsaI, not BsaI-HF (as described in the Golden Gate TALEN and TAL Effector Kit protocol) |
| BSA | New England Biolabs | provided with restriction enzymes | |
| 10x T4 ligase buffer | Promega (ordered through Fisher) | PR-C1263 | |
| GoTaq Green Master mix | Promega (ordered through Fisher) | PRM7123 | Other Taq can be used, but the reaction should be adjusted accordingly |
| Quick ligation kit | New England Biolabs (ordered through Fisher) | 50-811-728 | We use Quick Ligase for all TALEN assembly reactions |
| One Shot TOP10 Chemically Competent E.coli | Invitrogen | C4040-06 | Other chemically competent cells or homemade competent cells can be used |
| Esp 3I | Thermo Sci Fermentas Inc (Ordered through Fisher) | FERER0451 | |
| Plasmid-Safe ATP-dependent DNase | Epicentre (Ordered through Fisher) | NC9046399 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | The Qiagen kit should be used for the initial plasmid preparation (as described in the Golden Gate TALEN and TAL Effector Kit protocol) |
| QIAquick PCR Purification Kit | Qiagen | 28104 | |
| GeneMate LE Quick Dissolve Agaraose | BioExpress | E-3119-125 | |
| Sac I | Promega (Ordered through Fisher) | PR-R6061 | |
| mMESSAGE mMACHINE T3 Transcription kit | Ambion | AM1348M | |
| Rneasy MinElute Cleanup Kit | Qiagen | 74204 | |
| NorthernMax-Gly Sample Loading Dye | Ambion (ordered through Fisher) | AM8551 | |
| Eliminase | Decon (ordered through Fisher) | 04-355-32 | |
| Fisherbrand Disposable Soda-Lime Glass Pasteur Pipets | Fisher | 13-678-6B | |
| Standard Glass Capillaries | World Precision Instruments | 1B100-4 | |
| Microcaps | Drummond Scientific Company | 1-000-0010 | |
| Eppendorf Femtotips Microloader Tips for Femtojet Microinjector | Eppendorf (ordered through Fisher) | E5242956003 | |
| Sodium hydroxide | Fisher | S318-500 | |
| Tris base | Fisher | BP152-1 |
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