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

Este trabajo fue financiado en parte por el NIH R01 # DE021475 (CTM), una subvención del NIH predoctoral de Formación 5T32GM007127 (PAE), y una beca de investigación de Graduados de la NSF (NAE). Damos las gracias a Kevin Schwalbach para realizar recombinería y las inyecciones de BAC, Nick Donde para la generación de datos de secuenciación de Sanger CRISPR, y Katherine Lipari de información útil sobre el protocolo de inyección.

Los autores no tienen nada que revelar.

La inyección de embriones de espinosos de una sola célula para la transgénesis o la edición genoma presenta tres retos principales. En primer lugar, en relación con embriones de pez cebra, el pez espinoso embrionaria corion es difícil y, a menudo romper las agujas. Este problema puede superarse parcialmente mediante el uso de micropipetas de vidrio más gruesas y más fuertes y la inyección perpendicular al corion (véase el Protocolo, la Figura 2). Asegurar que tan poco agua como sea posible se ...

Un componente fundamental de la comprensión de cómo surge la biodiversidad es la determinación de las bases genéticas y de desarrollo de los cambios fenotípicos evolucionado en la naturaleza. El pez espinoso de tres espinas, Gasterosteus aculeatus, se ha revelado como un excelente modelo para estudiar la base genética de la evolución. Espinosos han sufrido muchos cambios evolutivos de adaptación como los peces marinos han colonizado un sinnúmero de ambientes de agua dulce en todo el hemisferio norte, lo que resulta en morfológica dramática, fisiológicos y cambios de conducta 1. Los genomas de individuos de veintiún poblaciones de espinosos se han secuenciado y ensamblado, y un mapa de ligamiento de alta densidad se ha generado para mejorar aún más el conjunto de 2,3. Experimentos de mapeo genéticos han identificado regiones genómicas subyacente fenotipos evolucionado 4-6, y en unos pocos casos, el papel funcional de genes candidatos específicos se han probado 7,8. Un número de regiones genómicas que generan cambios morfológicos se han identificado con prometedores genes candidatos, pero estos candidatos no han sido aún probado funcionalmente 9 - 12 El. Además, los espinosos son modelos comunes para estudios de genética de poblaciones / genómica 13,14, 15 especiación, comportamiento, endocrinología 1 16 17, ecotoxicología, la inmunología y parasitología 18 19. Los estudios futuros en cada uno de estos campos se beneficiarán de la capacidad de realizar manipulaciones genéticas funcionales en los espinosos. Además de la manipulación de sus secuencias de codificación, las funciones de los genes candidatos pueden evaluarse mediante el estudio de sus secuencias cis-regulador y aumentando funcionalmente, disminuyendo o eliminando la expresión del gen candidato. Métodos de microinyección y la transgénesis en los espinosos están bien establecidos 7,8,20 y fueron inicialmente desarrollados usando una meganucleasa mediada21 método descrito por primera vez en 22 medaka. El método de microinyección modificado que aquí se presenta ha sido optimizada tanto para la transgénesis mediada por Tol2 y recientemente desarrollado reactivos de edición del genoma incluyendo Talens y CRISPRs. Se cree que los cambios en los elementos cis-regulador a ser crítica para la evolución morfológica, como cis-regulador cambios pueden evitar las consecuencias negativas de las mutaciones pleiotrópicos 23 de codificación. Por lo tanto, las pruebas y la comparación de secuencias cis-regulador putativo se ha convertido en un objetivo central de un número creciente de estudios evolutivos. Además, la mayoría de las variantes de la enfermedad humanos son variantes de regulación 24,25 y sistemas modelo de vertebrados son muy necesarios para estudiar cis-regulador Función del elemento y la lógica. Los peces que fertilizar sus embriones externamente en un gran número de vertebrados ofrecen sistemas potentes para estudiar -Reglamento cis. El sistema de transposón Tol2, en el que foreign ADN de integrarse en el genoma está flanqueado por Tol2 transposasa sitios de unión y co-inyectados con ARNm transposasa Tol2, funciona con alta eficiencia para la integración con éxito plásmido construye en los genomas de peces 26 - 28. Típicamente, un promotor de potencial se clona aguas arriba de un promotor basal (como hsp70l 29) y el gen reportero fluorescente tal como EGFP (proteína fluorescente verde potenciada) o mCherry en una cadena principal de Tol2 y se inyecta con transposasa mRNA 26. La observación de la expresión del indicador fluorescente, ya sea en embriones o descendientes con transgenes integrados de forma estable inyectados, proporciona información acerca de la regulación espacial y temporal de la expresión génica impulsado por el potenciador putativo. En experimentos adicionales, potenciadores validados se pueden utilizar para conducir la sobreexpresión específica de tejido de genes de interés. Para el análisis de las regiones cis-regulador más grandes, de alta calidad genoma a gran insertobibliotecas IC mediante cromosomas artificiales bacterianos (BAC) se han construido tanto para los peces espinosos marinos y de agua dulce 30. Estos BAC se pueden recombineered para reemplazar un gen con un gen indicador fluorescente en el contexto de un grande (150 a 200 kb) región genómica 31. El indicador fluorescente se expresa entonces en un patrón espacio-temporal según lo determinado por las secuencias reguladoras dentro de la CAV. Para los estudios en los peces, los sitios Tol2 se pueden añadir a la BAC para facilitar la integración genómica 32,33. En etapas posteriores del desarrollo cuando la hibridación in situ es técnicamente difícil, la lectura fluorescente de la BAC se puede utilizar para estudiar los patrones de expresión de genes, como se ha demostrado para la proteína morfogenética ósea 6 stickleback (BMP6) 20. Además, los patrones de expresión fluorescentes en un individuo pueden ser rastreados a través del tiempo, que no se puede lograr con la hibridación in situ. BAC también se puede utilizar para añadir un additional copia de una región genómica para aumentar la dosis de un gen de interés. Para el estudio de la función génica, edición genoma es un campo en expansión explosiva que se puede utilizar para producir cambios dirigidos a secuencias genómicas en una amplia variedad de organismos 34. Transcripción nucleasas efectoras del tipo activador de (Talens) son nucleasas modulares, específicos de secuencia originalmente aisladas a partir de patógenos de plantas que pueden ser diseñados precisamente para unirse directamente a una secuencia genómica de elección y generar una doble hebra romper 35,36. Agrupados espaciadas regularmente repeticiones palindrómicas cortas (CRISPR) / sistemas CAS fueron encontrados originalmente en bacterias y usan una guía de ARN y la proteína Cas9 para generar una ruptura en una secuencia de ADN diana complementaria a la guía 37. La posterior reparación de la rotura de doble cadena creada por ambos Talens y CRISPRs menudo deja tras de sí una pequeña inserción o deleción, lo que puede alterar la función de la secuencia diana35-37. En los espinosos, Talens se han utilizado para alterar la expresión de genes por la orientación un promotor de 20, y ambos Talens y CRISPRs han producido con éxito mutaciones en las secuencias (datos no publicados) de codificación. Un protocolo detallado para la generación de CRISPRs para uso en el pez cebra se puede utilizar como una guía para desarrollar CRISPRs para los espinosos 38. Transgénicos y genoma experimentos de edición requieren introducción de ácidos nucleicos en un embrión de una célula recién fertilizado. Mediante la introducción de la herramienta de transgén o de edición de genoma temprano en el desarrollo, se maximiza el número de células hijas genéticamente manipulados en el embrión. embriones inyectados se criban entonces visualmente para fluorescencia o molecularmente examinados para modificaciones del genoma. Si las células que contribuyen a la línea germinal están dirigidos con éxito, el transgén o mutación se pueden transmitir a un subconjunto de la descendencia, incluso cuando la letalidad post-inyección es alta. Los peces pueden ser de mosaico o outcrossedentrecruzan y su descendencia tamiza para recuperar los alelos mutantes o un transgén integrado de forma estable de interés. Este protocolo describe métodos para introducir los transgenes y reactivos de edición del genoma en embriones de espinosos de una sola célula y el seguimiento de las modificaciones que darán éxito.

<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

El pez espinoso de tres espinas se ha convertido en un potente sistema para estudiar la base genética de una amplia variedad de características morfológicas, fisiológicas y fenotipos de comportamiento. Las notablemente diversos fenotipos que han evolucionado a medida que las poblaciones marinas se adaptan a un sinnúmero de ambientes de agua dulce, junto con la capacidad de cruzar formas marinas y de agua dulce, proporcionan un sistema de vertebrados rara en la que la genética puede ser utilizado para mapear las regiones genómicas que controlan rasgos evolucionado. Excelentes recursos genómicos ya están disponibles, lo que facilita la disección genética molecular de los cambios evolucionados. Mientras que los experimentos de mapeo generar listas de genes candidatos interesantes, se requieren manipulaciones genéticas funcionales para poner a prueba las funciones de estos genes. La regulación génica puede ser estudiado con plásmidos informadores transgénicos y BACs integrados en el genoma utilizando el sistema de transposasa Tol2. Funciones de los genes candidatos específicos y cis-elementos reguladores pueden evaluarse mediante la inducción específicamutaciones con TALEN y CRISPR / Cas9 reactivos edición genoma. Todos los métodos requieren la introducción de ácidos nucleicos en fertilizados embriones stickleback una sola célula, una tarea que se hizo difícil por el espesor corion de embriones de espinosos y la relativamente pequeña y delgada de blastómeros. Aquí, un protocolo detallado para la microinyección de los ácidos nucleicos en embriones de espinosos se describe para aplicaciones transgénicas y edición de genoma para estudiar la expresión génica y la función, así como técnicas para evaluar el éxito de la transgénesis y recuperar líneas estables.

Para transgenes de genes informadores que tienen potenciador de la actividad, la inyección con éxito dará lugar a la expresión específica, celular del transgén (Figura 4A, 4C). Peces inyectados pueden ser outcrossed para producir líneas estables (ejemplo de una línea estable de BAC se muestra en la Figura 4B). La inyección de ADN en embriones de espinosos típicamente resulta en la letalidad mucho mayor que el ARN solo. Es típi...

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

La microinyección de transgénesis y Genoma edición en tres espinas espinosos

Manipulaciones transgénicas y la edición del genoma son críticos para probar funcionalmente las funciones de los genes y elementos cis-regulador. Aquí se presenta un protocolo de microinyección detallado para la generación de modificaciones genómicas (incluyendo construcciones mediadas por Tol2 fluorescentes transgén reportero, Talens, y CRISPRs) para el modelo emergente de pescado, el espinoso de tres espinas.

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 vistas.  University of California, Berkeley.  Manipulaciones transgénicas y la edición del genoma son críticos para probar funcionalmente las funciones de los genes y elementos cis-regulador. Aquí se presenta un protocolo de microinyección detallado para la generación de modificaciones genómicas (incluyendo construcciones mediadas por Tol2 fluorescentes transgén reportero, Talens, y CRISPRs) para el modelo emergente de pescado, el espinoso de tres espinas. 

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.

Microinyección de la proteína CRISPR/Cas9 en bagre de canal, Ictalurus punctatus, embriones de Gene Editing

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

Investigación

Genética

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.

Microinyección de embriones y la técnica de trasplante para la manipulación del genoma de 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.

Generación de ratones modificados genéticamente a través de la microinyección de ovocitos

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

Biología del Desarrollo

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.

Microinyección de ARNm y oligonucleótidos antisentido Morpholino en embriones de pez cebra.

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

Biología

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.

Micromanipulación de la expresión génica en el cerebro de pez cebra adulto Uso Microinyección cerebroventricular de oligonucleótidos Morpholino

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

Neurociencias

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.

Microinyección de ADN en los oculares en embriones de Xenopus laevis e imágenes de gFP que expresan los árboles axonales ópticos en los renacuajos de Xenopus vivos

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.

Microinyección de cigoto para la creación de alelos de genes de casete knock-in y flox en ratones

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.

Edición genoma en<em&gt; Astyanax mexicanus</em&gt; Uso de transcripción activador de efector nucleasas (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.

Silenciar la chispa: Edición del genoma CRISPR/Cas9 en peces débilmente eléctricos

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.

Técnica de inyección de embriones para la edición de genes en la garrapata de patas negras, Ixodes scapularis

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