genom Düzenleme<em> Astyanax mexicanustan</emKullanma> Transkripsiyon Aktivatör benzeri Efektör Nükleazlar (Talens)
Summary
Gen hedefleme mutajenez genom düzenleme teknikleri kullanılarak organizmaların geniş bir mümkündür. Burada, Astyanax mexicanus, yüzey balık ve cavefish içeren balık türlerinin efektör nükleazlar (Talens) gibi transkripsiyon aktivatörü kullanılarak hedeflenen gen mutasyon için bir protokol göstermektedir.
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
Sürekli evrim genetik temelini anlamak evrimci biyologların kritik bir araştırma hedeftir. Önemli ilerleme özelliklerin evrimini altında yatan lokusların belirlenmesi ve (örneğin 1-3) bu loci içinde aday genlerin saptayarak yapılmamıştır. Ancak, fonksiyonel bu genlerin rolü özelliklerin evrimini incelemek için kullanılan birçok organizmaları zorlu kaldı etmiştir test şu anda genetik uysal değildir. Genom düzenleme teknolojileri çıkması büyük ölçüde organizmaların geniş bir genetik uygulanabılirliği artmıştır. Transkripsiyon aktivatörü gibi efektör nükleazlar (TALENS) ve kümelenmiş düzenli aralıklarla yerleştirilmişlerdir kısa yineleyen tekrarlar (CRISPRs) (Örnek 4-11 için) organizmadan genler hedefli mutasyon üretmek için kullanılmıştır. Bir evrimsel ilgili sisteme uygulanan bu araçları, evrimsel biyologlar evrimin genetik temeli çalışma yol devrim potansiyeline sahip.
Astyanax mexicanustan iki şekilde bulunur balık türüdür. Bir nehir yaşayan yüzey formu (yüzey balık) ve çoklu mağaralarda yaşayan formları (cavefish) A. mexicanustan cavefish (12 gözden) yüzey balık atalarından evrimleştiği. Cavefish popülasyonu gibi okullaşma davranışının kaybı, artan saldırganlık, duruş ve hiperfajya 13 beslenme değişiklikleri gibi davranış damak tadınızı ve kafatası neuromasts ve değişikliklerin sayısında artış, azalma ya da pigmentasyon kaybı, gözlerde kaybı da dahil olmak üzere özellikleri bir dizi geliştiğini -19. Cavefish ve yüzey balıkları interfertile, ve genetik haritalama deneyleri mağara özellikleri 1,20-26 için lokus ve aday genleri tanımlamak için yapılmıştır. Bazı aday genler diğer türlerin 21 model organizmalarda veya aşırı ifadesinin 27 veya geçici demonte u tarafından, hücre kültürü 1,19 mağara özellikleri katkıda işlevsel rolü için test edilmiştirA. morpholinos 28 şarkı mexicanustan. Bununla birlikte, bu yöntemlerin her biri, bir sınırlama vardır. A. Bu genlerin mutant alelleri oluşturma yeteneği mexicanustan cavefish evriminde işlevini anlamak için kritik önem taşır. Bu nedenle, A. mexicanustan genom düzenleme teknolojileri uygulanması için ideal bir aday organizmadır.
Burada A. genom düzenleme için bir yöntem özetlemektedir mexicanustan Talens kullanılmıştır. Bu yöntem, fenotip ve ilgi 29 genlerin kararlı mutasyonları ile balık hatları izole etmek için mozaik enjekte kurucu balık değerlendirmek için de kullanılabilir.
Protocol
Representative Results
Discussion
Büyük adımlar özelliklerin evrim genetik temelini anlamaya yönelik son yıllarda yapılmıştır. Özellikleri, bir dizi evrimi yatan aday gen belirlenmişse de, bunun nedeni en evrimsel ilginç türlerinin genetik tractability eksikliği, in vivo olarak, bu genlerin test etmek zor kalmıştır. Burada A. genom düzenleme için bir yöntem raporu mexicanustan, bir tür mağara hayvanların evrimi incelemek için kullandı. Genetik haritalama 1,21,23 çalışmaları ve aday gen …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Bu çalışma Genetik, Kalkınma ve Hücre Biyolojisi ve Iowa State Üniversitesi Bölümü tarafından ve NIH hibe EY024941 (WJ) .Dr tarafından finanse edildi. Jeffrey Essner el yazması üzerine yorum sağladı.
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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