Name: microinjection for transgenesis and genome editing in threespine sticklebacks
Uploaded: 2016-05-13T15:00:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

Questo lavoro è stato finanziato in parte dal NIH R01 # DE021475 (CTM), un NIH predoctoral formazione di Grant 5T32GM007127 (PAE), e un Graduate Research Fellowship NSF (NAE). Ringraziamo Kevin Schwalbach per l&#39;esecuzione di BAC recombineering e iniezioni, Nick Donde per la generazione di dati di sequenziamento Sanger CRISPR, e Katherine Lipari per il feedback utile sul protocollo di iniezione.

Gli autori non hanno nulla da rivelare.

Iniettano embrioni stickleback una cella per transgenesi o la modifica del genoma presenta tre sfide principali. In primo luogo, relativamente agli embrioni di zebrafish, lo spinarello embrionali corion è dura e spesso rompere gli aghi. Questo problema può essere superato parzialmente usando micropipette di vetro più spesse e più forti e iniettando perpendicolare al corion (vedi protocollo, figura 2). Assicurare che il meno acqua possibile si aggiunge agli embrioni (quanto basta per causare il corio...

Una componente fondamentale di capire come la biodiversità si pone è la determinazione delle basi genetiche e di sviluppo dei cambiamenti fenotipici evoluti in natura. Il pesce Spinarello, Gasterosteus aculeatus, è emerso come un ottimo modello per studiare le basi genetiche dell&#39;evoluzione. Spinarelli hanno subito molti cambiamenti evolutivi adattativi come i pesci marini hanno colonizzato gli ambienti di acqua dolce innumerevoli intorno nell&#39;emisfero settentrionale, con conseguente morfologica drammatico, fisiologici, e cambiamenti comportamentali 1. I genomi di individui di ventuno popolazioni stickleback sono stati sequenziati e assemblati, e una mappa linkage alta densità è stato generato per migliorare ulteriormente il gruppo 2,3. Esperimenti di mappatura genetici hanno identificato regioni genomiche sottostante fenotipi evoluti 4 - 6, e in alcuni casi, i ruoli funzionali di geni specifici sono stati testati 7,8. Un certo numero di regioni genomiche sottostanti cambiamenti morfologici sono stati identificati con promettenti geni candidati, ma questi candidati non sono ancora stati testati funzionalmente 9 - 12. Inoltre, spinarelli sono modelli comuni per gli studi di genetica delle popolazioni / genomica 13,14, speciazione 15, il comportamento 1, endocrinologia 16, ecotossicologia 17, l&#39;immunologia 18 e parassitologia 19. Futuri studi in ciascuno di questi settori beneficeranno della possibilità di eseguire manipolazioni genetiche funzionali in spinarelli. Oltre a manipolare le sequenze codificanti, i ruoli dei geni candidati possono essere valutate studiando le loro sequenze -regulatory cis e funzionalmente crescente, decrescente o eliminando espressione del gene candidato. Microiniezione e transgenesi metodi in spinarelli sono ben stabiliti 7,8,20 e sono stati inizialmente sviluppati utilizzando un meganuclease-mediataMetodo 21 descritto per primo nel Medaka 22. Il metodo microiniezione modificato presentato qui è stato ottimizzato sia per la transgenesi Tol2-mediata e recentemente sviluppato reagenti di modifica del genoma tra Talens e CRISPRs. Modifiche a cis -regulatory elementi sono pensati per essere critico per l&#39;evoluzione morfologica, come cis -regulatory cambiamenti possono evitare le conseguenze negative pleiotropici di codifica mutazioni 23. Pertanto, prova e il confronto putative sequenze -regulatory cis è diventato un obiettivo centrale di un numero crescente di studi evolutivi. Inoltre, la maggior parte delle varianti di malattie umane sono varianti normative 24,25 e sistemi modello vertebrati sono dolorosamente necessari per studiare cis Funzione di un elemento -regulatory e la logica. I pesci che fertilizzare i loro embrioni esternamente in gran numero offrono potenti sistemi di vertebrati a studiare -Regolamento cis. Il sistema di trasposoni Tol2, in cui commDNA gn per essere integrato nel genoma è fiancheggiato da Tol2 trasposasi siti di legame e di co-iniettato con Tol2 trasposasi mRNA, funziona con alta efficienza per l&#39;integrazione con successo plasmide costruisce sui genomi di pesce 26 - 28. In genere, un potenziale Enhancer è clonato a monte di un promotore basale (come hsp70l 29) e del gene reporter fluorescente come EGFP (enhanced green fluorescent protein) o mCherry in una spina dorsale Tol2 e iniettato con trasposasi mRNA 26. L&#39;osservazione di espressione del reporter fluorescente, sia in embrioni o discendenti con transgeni stabilmente integrati iniettati, fornisce informazioni sulla regolazione spazio-temporale dell&#39;espressione genica guidato dal enhancer putativo. In ulteriori esperimenti, esaltatori validati possono essere utilizzati per guidare sovraespressione tessuto-specifica dei geni di interesse. Per l&#39;analisi delle più grandi regioni -regulatory cis, di alta qualità genom grande insertolibrerie IC utilizzando cromosomi batterici artificiali (BAC) sono stati costruiti per entrambe le marine e di acqua dolce spinarelli 30. Questi BAC possono essere recombineered per sostituire un gene con un gene reporter fluorescente nel contesto di un grande (150-200 kb) regione genomica 31. Il reporter fluorescente viene poi espressa in un modello spazio-temporale, come determinato dal sequenze regolatrici all&#39;interno del BAC. Per gli studi nei pesci, siti Tol2 possono essere aggiunti alla BAC per facilitare genomico integrazione 32,33. Nelle fasi successive di sviluppo, quando l&#39;ibridazione in situ è tecnicamente impegnativo, la lettura fluorescenza del BAC può essere utilizzato per studiare gli schemi di espressione genica, come è stato dimostrato per stickleback proteina morfogenetica 6 (BMP6) 20. Inoltre, i modelli di espressione fluorescenti in un individuo possono essere monitorati nel corso del tempo, che non può essere realizzato con l&#39;ibridazione in situ. BAC può essere utilizzato anche per aggiungere un additional copia di una regione genomica di aumentare il dosaggio di un gene di interesse. Per lo studio della funzione del gene, editing genoma è un campo esplosivo espansione che può essere utilizzato per produrre cambiamenti mirati a sequenze genomiche in un&#39;ampia varietà di organismi 34. Trascrizione attivatore-come nucleasi effettrici (Talens) sono, nucleasi modulari sequenza-specifiche originariamente isolato da agenti patogeni delle piante che possono essere progettati proprio per legarsi direttamente ad una sequenza genomica di scelta e di generare un doppio filamento pausa 35,36. Cluster regolarmente intervallati brevi ripetizioni palindromi (CRISPR) / sistemi CAS sono stati originariamente trovati nei batteri e utilizzare un RNA guida e la proteina Cas9 per generare una pausa in una sequenza di DNA bersaglio complementare alla guida 37. La successiva riparazione della rottura a doppio filamento creato da entrambe le Talens e CRISPRs spesso lascia dietro di sé una piccola inserimento o la cancellazione, che possono interferire con la funzione della sequenza bersaglio35-37. In spinarelli, Talens sono stati utilizzati per distruggere l&#39;espressione genica di mira un potenziatore 20, ed entrambi Talens e CRISPRs hanno prodotto con successo le mutazioni in sequenze codificanti (dati non pubblicati). Un protocollo dettagliato per la generazione di CRISPRs per l&#39;utilizzo in zebrafish può essere utilizzato come linea guida per sviluppare CRISPRs per spinarelli 38. esperimenti di modifica transgenici e genoma richiedono l&#39;introduzione di acidi nucleici in un appena fecondato embrione unicellulare. Con l&#39;introduzione del transgene strumento o del genoma di modifica nelle prime fasi di sviluppo, il numero di cellule figlie geneticamente manipolate in l&#39;embrione è massimizzata. embrioni iniettati vengono poi proiettati visivamente per fluorescenza o molecolarmente sottoposti a screening per le modifiche del genoma. Se le cellule che contribuiscono alla linea germinale sono mirati con successo, il transgene o mutazioni possono essere trasmessi a un sottoinsieme di prole, anche quando post-iniezione letale è elevata. Il pesce a mosaico possono essere outcrossed oincrocia e la loro prole proiettato per recuperare i alleli mutanti o un transgene stabilmente integrata di interesse. Questo protocollo descrive i metodi per l&#39;introduzione di transgeni e l&#39;editing di reagenti genoma in embrioni spinarello uno-cellula e di monitoraggio per le modifiche genomiche di successo.

<table><tbody><tr><td>Stereomicroscope with transillumination</td><td>Leica</td><td>S6e/ KL300 LED</td><td></td></tr><tr><td>Manual micromanipulator</td><td>Applied Scientific Instrumentation</td><td>MM33</td><td>Marzhauser M33 Micromanipulator</td></tr><tr><td>Pressure Injecion system</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr><tr><td>Back pressure unit</td><td>Applied Scientific Instrumentation</td><td>BPU</td><td></td></tr><tr><td>Micropipette holder kit</td><td>Applied Scientific Instrumentation</td><td>MPIP</td><td></td></tr><tr><td>Magnetic base holder</td><td>Applied Scientific Instrumentation</td><td>Magnetic base</td><td></td></tr><tr><td>Foot switch</td><td>Applied Scientific Instrumentation</td><td>FSW</td><td></td></tr><tr><td>Iron plate (magnetic base)</td><td>Narishige</td><td>IP</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Disposable transfer pipettes</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>0.5% phenol red in DPBS</td><td>Sigma&amp;nbsp;</td><td>P0290</td><td>injection tracer</td></tr><tr><td>#5 forceps, biologie dumoxel&amp;nbsp;</td><td>Fine Science Tools</td><td>11252-30</td><td>for needle breaking</td></tr><tr><td>Micropipette Storage Jar</td><td>World Precision Instruments</td><td>E210</td><td>holds needles</td></tr><tr><td>6&quot;, 6 teeth per inch plaster drywall saw</td><td>Lenox</td><td>20571 (S636RP)</td><td>hold eggs for injection</td></tr><tr><td>13 cm x 13 cm glass plate</td><td>any hardware store</td><td>-</td><td></td></tr><tr><td>Borosilicate glass capillaries, 1.0 mm OD/0.58 mm ID&amp;nbsp;</td><td>World Precision Instruments</td><td>1B100-F4</td><td>*harder glass than zebrafish injection capillaries</td></tr><tr><td>150 x 15mm Petri dish</td><td>Fisher</td><td>FB0875714</td><td>raise stickleback embryos</td></tr><tr><td>35 mm x 10 mm Petri dish</td><td>Fisher</td><td>08-757-100A</td><td>store eggs pre-injection</td></tr><tr><td>Instant Ocean Salt</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>Tricaine methanesulfonate/MS-222</td><td>Western Chemical Inc</td><td>MS222</td><td>fish anaesthesia/euthanasia</td></tr><tr><td>Sp6 transcription kit</td><td>Ambion</td><td>AM1340</td><td>For transcription of TALENs and transposase mRNA</td></tr><tr><td>RNeasy cleanup kit</td><td>Qiagen</td><td>74104</td><td>purify transposase or TALEN RNA</td></tr><tr><td>QiaQuick PCR cleanup kit</td><td>Qiagen</td><td>28104</td><td>clean up plasmids for injection</td></tr><tr><td>Proteinase K 20 mg/ml</td><td>Ambion</td><td>AM2546</td><td>for DNA preparation</td></tr><tr><td>Nucleobond BAC 100 kit</td><td>Clontech</td><td>740579</td><td>for BAC DNA preparation</td></tr><tr><td>NotI</td><td>NEB</td><td>R0189L</td><td></td></tr><tr><td>Phusion polymerase</td><td>Fisher</td><td>F-530L</td><td></td></tr><tr><td>Qiagen PlasmidPlus Midi kit</td><td>Qiagen</td><td>12943</td><td>contains endotoxin rinse buffer</td></tr><tr><td>QIAQuick Gel Extraction</td><td>Qiagen</td><td>28704&amp;nbsp;</td><td>for sequencing induced mutations</td></tr><tr><td>Phenol:chloroform:Isoamyl alcohol</td><td>Sigma</td><td>P2069-100ML</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>S2889-250G</td><td></td></tr><tr><td>Ethanol (molecular biology grade)</td><td>Sigma</td><td>E7023-500ML</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9539</td><td></td></tr><tr><td>50x Tris-acetate-EDTA buffer</td><td>ThermoFisher</td><td>B49</td><td></td></tr><tr><td>0.5-10 Kb RNA ladder</td><td>ThermoFisher</td><td>15623-200</td><td></td></tr><tr><td>Nanodrop&amp;nbsp; Spectrophotometer</td><td>Thermo Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127-500G</td><td></td></tr><tr><td>10x PBS</td><td>ThermoFisher</td><td>70011-044</td><td></td></tr><tr><td>1 kb Plus DNA Ladder</td><td>ThermoFisher</td><td>10787-018</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9541-500G</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M8266-100G</td><td></td></tr><tr><td>NP-40</td><td>ThermoFisher</td><td>28324</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P1379-500ML</td><td></td></tr><tr><td>Tris pH 8.3</td><td>Teknova</td><td>T1083</td><td></td></tr><tr><td>12-strip PCR tube</td><td>Thermo Scientific</td><td>AB-1113</td><td></td></tr></tbody></table>

microinjection for transgenesis and genome editing in threespine sticklebacks

Il pesce Spinarello è emersa come un potente sistema per studiare la base genetica di una vasta gamma di morfologica, fisiologica, e fenotipi comportamentali. I fenotipi notevolmente diverse che si sono evoluti come le popolazioni marine si adattano agli ambienti di acqua dolce innumerevoli, combinata con la capacità di attraversare le forme marine e d&#39;acqua dolce, forniscono un sistema di vertebrati raro in cui la genetica può essere utilizzato per mappare regioni genomiche il controllo sia evoluto tratti. Eccellenti risorse genomiche sono ora disponibili, facilitando molecolare dissezione genetica dei cambiamenti evoluti. Mentre gli esperimenti di mappatura generano liste di geni candidati interessanti, manipolazioni genetiche funzionali sono necessari per testare il ruolo di questi geni. regolazione genica può essere studiato con plasmidi reporter transgenici e BAC integrate nel genoma utilizzando il sistema trasposasi Tol2. Funzioni di specifici geni candidati e gli elementi -regulatory cis possono essere valutate inducendo miratamutazioni con Talen e CRISPR / Cas9 reagenti modifica del genoma. Tutti i metodi richiedono l&#39;introduzione di acidi nucleici in fecondate embrioni spinarello un cellulare, un compito reso difficile dalla fitta corion di embrioni stickleback e il relativamente piccolo e sottile blastomeri. Qui, un protocollo dettagliato per la microiniezione di acidi nucleici in embrioni spinarello è descritto per le applicazioni transgeniche e la modifica del genoma per studiare l&#39;espressione genica e la funzione, così come le tecniche per valutare il successo di transgenesi e recuperare le linee stabili.

Per transgeni Reporter gene che hanno attività enhancer, iniezione di successo si tradurrà in specifico, espressione cellulare del transgene (Figura 4A, 4C). Pesci iniettati possono essere outcrossed per produrre linee stabili (esempio di una linea stabile BAC mostrato nella Figura 4B). L&#39;iniezione di DNA in embrioni stickleback in genere si traduce in letalità di gran lunga superiore rispetto alla sola RNA. È tipico vedere fino ...

Watch this Scientific Journal Video about Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks at JoVE.com

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Microiniezione per Transgenesi e Genome Editing in Threespine spinarelli

Manipolazioni transgeniche e la modifica del genoma sono fondamentali per il collaudo funzionale i ruoli dei geni ed elementi -regulatory cis. Ecco una dettagliata protocollo microiniezione per la generazione di modifiche genomiche (compresi costrutti Tol2-mediate fluorescenti giornalista transgene, Talens, e CRISPRs) è presentato per il pesce modello emergente, il Spinarello.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

microinjection-for-transgenesis-genome-editing-threespine

Research

JoVE Journal

Developmental Biology

13.9K Views.  University of California, Berkeley.  Manipolazioni transgeniche e la modifica del genoma sono fondamentali per il collaudo funzionale i ruoli dei geni ed elementi -regulatory cis. Ecco una dettagliata protocollo microiniezione per la generazione di modifiche genomiche (compresi costrutti Tol2-mediate fluorescenti giornalista transgene, Talens, e CRISPRs) è presentato per il pesce modello emergente, il Spinarello. 

Video: Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

Microinjection di proteine CRISPR/Cas9 in Channel Catfish, Ictalurus punctatus, embrioni per Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Ricerca

Genetica

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

Microinjection di embrione e tecnica di trapianto per la manipolazione del genoma di Nasonia vitripennis

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

Generazione di topi modificati geneticamente attraverso la microinjection of oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Biologia dello sviluppo

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microiniezione di mRNA e oligonucleotidi antisenso Morpholino in embrioni di zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Biologia

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Micromanipolazione di espressione genica in Zebrafish adulti cervello con microiniezione Cerebroventricular di Morpholino oligonucleotidi

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Neuroscienze

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microiniezione di DNA in boccioli di occhiali in embrioni di Xenopus lavis e imaging di GFP Expressing Optic Aboral Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

Microiniezione di zigote per la creazione di alleli knock-in e flox a cassetta genica nei topi

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

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.

Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases TALENs

Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish.

Genome Editing in<em&gt; Mexicanus Astianatte</em&gt; Uso Trascrizione Activator simile Effector nucleasi (Talens)

Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs.  Towards this end, much recent ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Silenziamento della scintilla: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Tecnica di iniezione di embrioni per l'editing genetico nella zecca dalle zampe nere, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...