Genome redigering i<em> Astyanax mexicanus</em> Använda transkriptionsaktivator-liknande Effector nukleaser (Talens)
Summary
Gene inriktning mutagenes är nu möjligt i ett brett spektrum av organismer med genomet redigeringstekniker. Här visar vi ett protokoll för målinriktad gen mutagenes med användning transkriptionsaktivator som effektor nukleaser (Talens) i Astyanax mexicanus, en fiskart som omfattar yta fisk och cavefish.
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
Att förstå den genetiska grunden för drag utveckling är en kritisk forskning mål av evolutionsbiologer. Betydande framsteg har gjorts när det gäller att identifiera loci ligger till grund för utvecklingen av egenskaper och lokalisera kandidatgener inom dessa loci (t.ex. 1-3). Men funktionellt testa rollen av dessa gener har förblivit utmanande många organismer som används för att studera utvecklingen av egenskaper är för närvarande inte genetiskt lätthanterlig. Tillkomsten av genomet redigeringstekniker har i hög grad ökad genetisk manipulability av ett brett spektrum av organismer. Transkriptionsaktivator liknande effektor nukleaser (Talens) och klustrade regelbundet mellanrum korta palindromiska upprepningar (CRISPRs) har använts för att generera riktade mutationer i gener i ett antal organismer (till exempel 4-11). Dessa verktyg, som tillämpas på ett evolutionärt relevanta systemet har potential att revolutionera hur evolutionsbiologer studera den genetiska grunden för evolutionen.
Astyanax mexicanus är en fiskart som finns i två former: a. Flod bostad yta formen (ytan fisk) och flera grottlevande former (cavefish) A. mexicanus cavefish utvecklats från ytan fisk förfäder (översikt i 12). Populationer av cavefish har utvecklats ett antal egenskaper, inklusive förlust av ögon, minska eller förlust av pigmentering, ökat antal smaklökar och kraniala neuromasts, och förändringar i beteende såsom förlust av skolbeteende, ökad aggressivitet, förändringar i utfodring hållning och hyperfagi 13 -19. Cavefish och ytan fisk är interfertile, och genetiska kartläggningsexperiment har utförts för att identifiera loci och kandidatgener för grottdrag 1,20-26. Vissa gener har testats för en funktionell roll för att bidra till grott egenskaper i cellodling 1,19, i modellorganismer av andra arter 21 eller genom överuttryck 27 eller övergående knockdown usjunga morpholinos 28 i A. mexicanus. Emellertid har var och en av dessa metoder begränsningar. Förmågan att generera mutanta alleler av dessa gener i A. mexicanus är avgörande för att förstå deras funktion i utvecklingen av cavefish. Således, A. mexicanus är en idealisk kandidat organism för tillämpning av genomet redigering teknik.
Här redogör vi en metod för genom redigering i A. mexicanus använder Talens. Denna metod kan användas för att utvärdera mosaik injiceras grundare fisk för fenotyper och för att isolera rader av fisk med stabila mutationer i gener av intresse 29.
Protocol
Representative Results
Discussion
Stora framsteg har gjorts under de senaste åren mot att förstå den genetiska grunden för utvecklingen av egenskaper. Även kandidatgener ligger till grund för utvecklingen av ett antal egenskaper har identifierats, har det varit en utmaning att testa dessa gener in vivo på grund av bristen på genetisk spårbarhet mest evolutionärt intressanta arter. Här rapporterar vi en metod för genomet redigering i A. mexicanus, en art som används för att studera utvecklingen av grott djur. Genetisk kart…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Detta arbete har finansierats av Institutionen för genetik, utveckling och cellbiologi och Iowa State University och av NIH bidrag EY024941 (WJ) .Dr. Jeffrey Essner lämnade synpunkter på manuskriptet.
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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