Genome bewerken in<em> Astyanax mexicanus</em> Met behulp van transcriptie Activator-achtige Effector Nucleases (Talens)
Summary
-Gen-targeting mutagenese is nu mogelijk in een breed scala van organismen, waarbij genoom montagetechnieken. Hier tonen we een protocol voor gerichte gen mutagenese met behulp van transcriptie-activator, zoals effector nucleasen (Talens) in Astyanax mexicanus, een soort vis dat oppervlak vis en cavefish omvat.
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
Inzicht in de genetische basis van trait evolutie een kritisch onderzoek doel van evolutionair biologen. Er is aanzienlijke vooruitgang geboekt bij het identificeren loci grondslag liggen aan de evolutie van de eigenschappen en het opsporen van kandidaat-genen binnen deze loci (bijvoorbeeld 1-3). Echter functioneel testen van de rol van deze genen is gebleven uitdagend veel organismen gebruikt voor het bestuderen van de ontwikkeling van eigenschappen momenteel niet genetisch handelbaar. De komst van het genoom editing technologieën is sterk toegenomen genetische maakbaarheid van een breed scala van organismen. Transcriptie activator-achtige effector nucleasen (Talens) en geclusterde regelmatig interspaced korte palindroom repeats (CRISPRs) gebruikt om gerichte mutaties in genen genereren verschillende organismen (bijvoorbeeld 4-11). Deze tools, toegepast op een evolutionair relevante systeem, hebben het potentieel om een revolutie in de manier waarop evolutionair biologen onderzoek naar de genetische basis van de evolutie.
Astyanax mexicanus is een vissoort die in twee vormen bestaat:. A-river woning oppervlak vorm (oppervlak vis) en multiple-grotwoning formulieren (cavefish) A. mexicanus cavefish ontstaan uit het oppervlak vis voorouders (besproken in 12). Populaties van cavefish hebben een aantal eigenschappen, waaronder het verlies van de ogen, te verminderen of verlies van pigmentatie geëvolueerd, verhoogde aantallen smaakpapillen en craniale neuromasts, en veranderingen in het gedrag, zoals het verlies van het onderwijs gedrag, toegenomen agressie, veranderingen in het voeden van houding en hyperfagie 13 -19. Cavefish en oppervlakte vissen zijn interfertile, en genetische mapping experimenten zijn uitgevoerd om loci en kandidaat-genen te identificeren voor grot trekken 1,20-26. Kandidaatgenen zijn getest voor een functionele rol bij hol eigenschappen in celkweek 1,19, in modelorganismen andere soorten 21 of 27 overexpressie of transiënte knockdown uzingen morpholinos 28 in A. mexicanus. Echter, elk van deze methoden heeft beperkingen. Het vermogen om gemuteerde allelen van deze genen in A. genereren mexicanus is essentieel voor het begrijpen van de functie in de evolutie van cavefish. Aldus A. mexicanus is een ideale kandidaat organisme voor de toepassing van genoom editing technologieën.
Hier schetsen we een methode voor het genoom bewerking in A. mexicanus gebruik TALENS. Deze methode kan worden gebruikt om mozaïek geïnjecteerde grondlegger vis evalueren fenotypes en voor het isoleren van leidingen vis met stabiele mutaties in genen van belang 29.
Protocol
Representative Results
Discussion
Grote vooruitgang is geboekt in de afgelopen jaren naar het begrijpen van de genetische basis van de evolutie van eigenschappen. Terwijl kandidaat genen voor de ontwikkeling van een aantal eigenschappen is geïdentificeerd, is het een uitdaging om deze genen in vivo testen gebleven door het gebrek aan volgzaamheid van genetische evolutionair meest interessante soorten. Hier melden wij een methode voor het genoom bewerking in A. mexicanus, een soort die wordt gebruikt om de evolutie van de grot dieren t…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk werd gefinancierd door de afdeling Genetica, Ontwikkeling en Celbiologie en Iowa State University en door NIH-subsidie EY024941 (WJ) .Dr. Jeffrey Essner verstrekt commentaar op het manuscript.
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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